Compounds, compositions, and methods

ABSTRACT

Compounds, compositions and methods useful for treating cellular proliferative diseases and disorders, for example, by modulating the activity of KSP, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of co-pending provisional U.S.Application Ser. No. 60/398,224, filed Jul. 23, 2002 incorporated hereinby reference.

FIELD OF THE INVENTION

This invention relates to quinazolinone-like derivatives that areinhibitors of the mitotic kinesin KSP and are useful in the treatment ofcellular proliferative diseases, for example cancer, hyperplasias,restenosis, cardiac hypertrophy, immune disorders and inflammation.

BACKGROUND OF THE INVENTION

The mitotic spindle is responsible for distribution of replicate copiesof the genome to each of the two daughter cells that result from celldivision. Disruption of the mitotic spindle can inhibit cell division,and induce cell death. Microtubules are the primary structural elementof the mitotic spindle; they are the site of action of certain existingtherapeutic agents used to treat cancer, such as taxanes and vincaalkaloids. Microtubules, however, exist as elements in other types ofcellular structures (including tracks for intracellular transport innerve processes). The therapeutic targeting of microtubules can,therefore, modulate processes in addition to cellular proliferation,leading to side effects that limit the usefulness of such drugs.

Improvement in the specificity of agents used to treat cancer is ofconsiderable interest because of the therapeutic benefits that would berealized if the side effects associated with the administration of theseagents could be reduced. Dramatic improvements in the treatment ofcancer have been associated with identification of therapeutic agentsacting through novel mechanisms. Examples of this include not only thetaxanes, but also the camptothecin class of topoisomerase I inhibitors.

One novel anti-proliferative mechanism entails selective inhibition ofmitotic kinesins, enzymes that are essential for assembly and functionof the mitotic spindle, but are not generally part of other microtubulestructures, such as in nerve processes. See, e.g., Guidebook to theCytoskeletal and Motor Proteins, Kreis and Vale, Eds., pp. 389-394(Oxford University Press 1999). Mitotic kinesins play essential rolesduring all phases of mitosis. These enzymes are “molecular motors” thattransform energy released by hydrolysis of ATP into mechanical forcethat drives the directional movement of cellular cargoes alongmicrotubules. The catalytic domain sufficient for this task is a compactstructure of approximately 340 amino acids. During mitosis, kinesinsorganize microtubules into the bipolar structure that is the mitoticspindle. Kinesins mediate movement of chromosomes along spindlemicrotubules, as well as structural changes in the mitotic spindleassociated with specific phases of mitosis. Experimental perturbation ofmitotic kinesin function causes malformation or dysfunction of themitotic spindle, frequently resulting in cell cycle arrest and celldeath. Mitotic kinesins are attractive targets for the discovery anddevelopment of novel anti-mitotic chemotherapeutics.

Among the mitotic kinesins that have been identified is KSP. KSP belongsto an evolutionarily conserved kinesin subfamily of plus end-directedmicrotubule motors that assemble into bipolar homotetramers consistingof antiparallel homodimers. During mitosis, KSP associates withmicrotubules of the mitotic spindle. Microinjection of antibodiesdirected against KSP into human cells prevents spindle pole separationduring prometaphase, giving rise to monopolar spindles and causingmitotic arrest and induction of programmed cell death. KSP and relatedkinesins in other, non-human, organisms, bundle antiparallelmicrotubules and slide them relative to one another, thus forcing thetwo spindle poles apart. KSP may also mediate in anaphase B spindleelongation and focussing of microtubules at the spindle pole.

Human KSP (also termed HsEg5) has been described [Blangy, et al., Cell,83:1159-69 (1995); Whitehead, et al., Arthritis Rheum., 39:1635-42(1996); Galgio et al., J. Cell Biol., 135:339-414 (1996); Blangy, etal., J Biol. Chem., 272:19418-24 (1997); Blangy, et al., Cell Motil.Cytoskeleton, 40:174-82 (1998); Whitehead and Rattner, J. Cell Sci.,111:2551-61 (1998); Kaiser, et al., JBC 274:18925-31 (1999); GenBankaccession numbers: X85137, NM004523 and U37426], and a fragment of theKSP gene (TRIP5) has been described [Lee, et al., Mol. Endocrinol.,9:243-54 (1995); GenBank accession number L40372]. Xenopus KSP homologs(Eg5), as well as Drosophila KLP61 F/KRP1 30 have been reported.

Recently, certain substituted quinazolinones have been described asinhibitors of mitotic kinesins for the treatment of cellularproliferative diseases (WO 01/30768 and WO 01/98278). It is an object ofthe present invention to provide novel inhibitors of mitotic kinesinssuch as KSP (particularly human KSP).

SUMMARY OF THE INVENTION

The present invention provides compounds, compositions and methodsuseful in the inhibition of mitotic kinesins, particularly KSP (moreparticularly human KSP). The compounds can be used to treat cellularproliferative diseases and include certain pyrrolidin-2-one,piperidin-2-one and tetrahydro-pyrimidin-2-one quinazolinonederivatives.

In one aspect, the invention relates to one or more compounds selectedfrom the group represented by Formula I:

where:

-   -   V is chosen from a covalent bond, CR′R″ and NR′″,        -   R′ and R″ being independently chosen from hydrogen, hydroxy,            optionally substituted aryl, optionally substituted            heteroaryl, optionally substituted amino, optionally            substituted alkyl and optionally substituted alkoxy, and        -   R′″ being chosen from hydrogen, optionally substituted            alkyl, optionally substituted aryl, optionally substituted            aralkyl, optionally substituted heteroaryl, and optionally            substituted heteroaralkyl;    -   R¹, R², R³ and R⁴ are independently chosen from hydrogen,        hydroxy, optionally substituted alkyl, optionally substituted        alkoxy, halogen, nitro, optionally substituted amino,        alkylsulfonyl, alkylsulfonamido, alkylsulfanyl, alkoxycarbonyl,        aminocarbonyl, optionally substituted aryl, optionally        substituted heteroaryl, and cyano;    -   R⁵ is chosen from hydrogen, optionally substituted alkyl,        optionally substituted aryl, optionally substituted aralkyl,        optionally substituted heteroaryl, and optionally substituted        heteroaralkyl;    -   R⁶ to R⁹ are independently chosen from hydrogen, hydroxy,        optionally substituted alkyl, optionally substituted alkoxy,        optionally substituted aryl, and optionally substituted        alkylamino, provided that neither R⁸ nor R⁹ is hydroxy or alkoxy        when V is NR′″; and    -   R¹⁰ is chosen from hydrogen, optionally substituted alkyl,        optionally substituted aryl, optionally substituted aralkyl,        optionally substituted heteroaryl, and optionally substituted        heteroaralkyl;        including single stereoisomers, mixtures of stereoisomers, and        pharmaceutically acceptable salts, solvates, and solvates of        pharmaceutically acceptable salts thereof. Compounds of Formula        I and pharmaceutically acceptable salts and solvates thereof are        useful as active agents in the practice of the methods of        treatment and in manufacture of compositions including the        pharmaceutical formulations of the invention, and may also be        useful as intermediates in the synthesis of such active agents.

In another aspect, the invention relates to one or more compoundsselected from the group represented by Formula II:

where:

-   -   T is a covalent bond or optionally substituted lower alkylene;    -   W, X, Y and Z are independently N, C, O, S or absent, provided        that:        -   no more than one of W, X, Y or Z is absent,        -   no more than two of W, X, Y and Z are —N═, and        -   W, X, Y or Z can be O or S only when one of W, X, Y or Z is            absent; and    -   R¹ to R¹⁰ and V are as defined with regard to Formula I,        provided that R¹, R², R³ or R⁴ is absent where W, X, Y or Z,        respectively, is —N═, O, S or absent;        including single stereoisomers, mixtures of stereoisomers, and        pharmaceutically acceptable salts, solvates, and solvates of        pharmaceutically acceptable salts thereof. The compounds        encompassed by Formula II and pharmaceutically acceptable salts        and solvates thereof will be seen to include those of Formula I;        they are likewise useful as active agents in the practice of the        methods of treatment and in manufacture of compositions        including the pharmaceutical formulations of the invention, and        may also be useful as intermediates in the synthesis of such        active agents.

In one of its particular aspects the present invention pertains to acompound represented by Formula I or II, having a substituent selectedfrom one or more of the following for R¹ to R⁴; R⁵; R⁶ to R⁹; R¹⁰; T; V;or W, X, Y and Z:

-   -   R¹, R², R³ and R⁴ are independently chosen from hydrogen, halo        (particularly chloro and fluoro), lower alkyl (particularly        methyl), substituted lower alkyl, lower alkoxy (particularly        methoxy), and cyano;    -   R⁵ is aralkyl or substituted aralkyl (particularly benzyl or        substituted benzyl; most particularly benzyl);    -   R⁶ to R⁹ are hydrogen;    -   R¹⁰ is optionally substituted benzyl or optionally substituted        phenyl (particularly tolylmethyl);    -   T is a covalent bond;    -   V is CH₂, N(H) or N(optionally substituted alkyl); and    -   W, X, Y and Z are —C═.        Other particular aspects of the invention pertain to methods and        to pharmaceutical formulations employing such a compound.

In one aspect, the invention relates to methods for treating cellularproliferative diseases, for disorders that can be treated by modulatingKSP kinesin activity, and for inhibiting KSP kinesin by theadministration of a therapeutically effective amount of a compound ofFormula I or II, or a pharmaceutically acceptable salt or solvate ofsuch compounds. Diseases and disorders that respond to therapy withcompounds of the invention include cancer, hyperplasia, restenosis,cardiac hypertrophy, immune disorders and inflammation.

In another aspect, the invention relates to a pharmaceutical compositioncontaining a therapeutically effective amount of a compound of Formula Ior II or a pharmaceutically acceptable salt or solvate thereof admixedwith at least one pharmaceutically acceptable excipient.

Yet another aspect of the invention relates to a kit having a compound,pharmaceutically acceptable salt or solvate of Formula I or II and apackage insert or other labeling including directions for treating acellular proliferative disease by administering an effective amount ofthe compound, salt or solvate. In one particular such aspect, thecompound, pharmaceutically acceptable salt or solvate of Formula I or IIis provided as a pharmaceutical composition.

In an additional aspect, the present invention provides methods ofscreening for compounds that will bind to a KSP kinesin, for examplecompounds that will displace or compete with the binding of thecompounds of the invention. The methods entail combining a labeledcompound of the invention, a KSP kinesin, and at least one candidateagent and determining the binding of the candidate agent to the KSPkinesin.

In a further aspect, the invention provides methods of screening formodulators of KSP kinesin activity. The methods entail combining acompound of the invention, a KSP kinesin, and at least one candidateagent and determining the effect of the candidate agent on the KSPkinesin activity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds, compositions and methodsuseful in the inhibition of mitotic kinesins, particularly KSP (moreparticularly human KSP). The compounds can be used to treat cellularproliferative diseases and include certain pyrrolidin-2-one,piperidin-2-one and tetrahydro-pyrimidin-2-one quinazolinonederivatives. The invention further relates to pharmaceuticalformulations comprising compounds of the invention, and methods oftreatment employing such compounds or compositions.

Definitions

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise. The following abbreviations and terms have the indicatedmeanings throughout:

Ac=acetyl

Boc=t-butyloxy carbonyl

Bu=butyl

c-=cyclo

CBZ=carbobenzoxy=benzyloxycarbonyl

DCM=dichloromethane=methylene chloride=CH₂Cl₂

DIEA=N,N-diisopropylethylamine

DMF=N,N-dimethylformamide

DMSO=dimethyl sulfoxide

Et=ethyl

Me=methyl

rt=room temperature

s-=secondary

t-=tertiary

TFA=trifluoroacetic acid

THF=tetrahydrofuran

The substituents identified as V, W and Y are intended to have themeanings set forth in the Summary, this Detailed Description and theClaims; they are not intended to designate the atomic elements Vanadium,Tungsten and Yttrium.

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. For example, “optionally substitutedalkyl” includes “alkyl” and “substituted alkyl,” as defined below. Itwill be understood by those skilled in the art with respect to any groupcontaining one or more substituents that such groups are not intended tointroduce any substitution or substitution patterns that are stericallyimpractical, synthetically non-feasible and/or inherently unstable.

“Alkyl” is intended to include linear, branched, or cyclic aliphatichydrocarbon structures and combinations thereof, which structures may besaturated or unsaturated (particularly having up to 20 carbon atoms,more particularly up to C₁₃.). Lower alkyl refers to alkyl groups offrom 1 to 5 (particularly 1 to 4) carbon atoms. Examples of lower alkylgroups include methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyland the like. Cycloalkyl is a subset of alkyl and includes cyclicaliphatic hydrocarbon groups of from 3 to 13 carbon atoms. Examples ofcycloalkyl groups include c-propyl, c-butyl, c-pentyl, norbornyl,adamantyl and the like. In this application, alkyl refers to alkanyl,alkenyl and alkynyl residues; it is intended to includecyclohexylmethyl, vinyl, allyl, isoprenyl and the like. Alkylene,alkenylene and alkynylene are other subsets of alkyl, referring to thesame residues as alkyl, but having two points of attachment. Examples ofalkylene include ethylene (—CH₂CH₂—), ethenylene (—CH═CH—), propylene(—CH₂CH₂CH₂—), dimethylpropylene (—CH₂C(CH₃)₂CH₂—) andcyclohexylpropylene (—CH₂CH₂CH(C₆H₁₃)—). When an alkyl residue having aspecific number of carbons is named, all geometric isomers of thatresidue having the specified number of carbons are meant to be included;thus, for example, “butyl” is meant to include n-butyl, sec-butyl,isobutyl and t-butyl; “propyl” includes n-propyl and isopropyl.

The term “alkoxy” or “alkoxyl” refers to the group —O-alkyl,particularly including from 1 to 8 carbon atoms in a straight, branchedor cyclic configuration, or combinations thereof, attached to the parentstructure through an oxygen. Examples include methoxy, ethoxy, propoxy,isopropoxy, cyclopropyloxy, cyclohexyloxy and the like. Lower-alkoxyrefers to groups containing one to five carbons.

The term “substituted alkoxy” refers to the group —O-(substitutedalkyl). One particular substituted alkoxy group is “polyalkoxy” or—O-(optionally substituted alkylene)-(optionally substituted alkoxy),and includes groups such as —OCH₂CH₂OCH₃, and glycol ethers such aspolyethyleneglycol and —O(CH₂CH₂O)_(x)CH₃, where x is an integer ofabout 2-20, particularly about 2-10, and more particularly about 2-5.Another particular substituted alkoxy group is hydroxyalkoxy or—OCH₂(CH₂)_(y)OH, where y is an integer of about 1-10, particularlyabout 1-4.

“Acyl” refers to groups of from 1 to 8 carbon atoms in a straight,branched or cyclic configuration, or combinations thereof, or to ahydrogen atom attached to the parent structure through a carbonylfunctionality. Such groups may be saturated or unsaturated, andaliphatic or aromatic. One or more carbons in the acyl residue may bereplaced by nitrogen, oxygen or sulfur as long as the point ofattachment to the parent remains at the carbonyl. Examples includeformyl, acetyl, benzoyl, propionyl, isobutyryl, t-butoxycarbonyl,benzyloxycarbonyl, aminocarbonyl, and the like. Lower-acyl refers to anacyl group containing one to five carbons. “Substituted acyl” refers toan acyl group where one or more of the hydrogens otherwise attached to acarbon, nitrogen or sulfur atom is substituted, the point of attachmentto the parent moiety remaining at the carbonyl.

The term “acyloxy” refers to the group —O-acyl. “Substituted acyloxy”refers to the group —O-substituted acyl.

The term “amidino” refers to the group —C(═NH)—NH₂. The term“substituted amidino” refers to the formula —C(═NR)—NRR in which each Ris independently selected from the group: hydrogen, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedaminocarbonyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted heterocyclyl, acyl, alkoxycarbonyl,sulfanyl, sulfinyl and sulfonyl, provided that at least one R is nothydrogen.

The term “amino” refers to the group —NH₂. The term “substituted amino”refers to the group —NHR or —NRR where each R is independently selectedfrom the group: optionally substituted acyl, optionally substitutedalkyl, optionally substituted alkoxy, optionally substituted amino,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocyclyl, sulfinyl and sulfonyl, e.g.,methylamino, dimethylamino, diethylamino, methylsulfonylamino,furanyl-oxy-sulfonamino, guanidino.

“Aryl” and “heteroaryl” mean a 5- or 6-membered aromatic ring orheteroaromatic ring containing 1-4 heteroatoms selected from O, N, or S;a bicyclic 9- or 10-membered aromatic ring system or heteroaromatic ringsystem containing 1-4 (or more) heteroatoms selected from O, N, or S; ora tricyclic 13- or 14-membered aromatic ring system or heteroaromaticring system containing 1-4 (or more) heteroatoms selected from O, N, orS. The aromatic 6- to 14-membered carbocyclic rings include, e.g.,benzene, naphthalene, indane, tetralin, and fluorene and the 5- to10-membered aromatic heterocyclic rings include, e.g., imidazole,pyridine, indole, thiophene, benzopyranone, thiazole, furan,benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine,pyrazine, tetrazole and pyrazole; particularly imidazole andimidazoline.

“Aralkyl” refers to a residue in which an aryl moiety is attached to theparent structure via an alkyl residue. Examples include benzyl,phenethyl, phenylvinyl, phenylallyl and the like. “Heteroaralkyl” refersto a residue in which a heteroaryl moiety is attached to the parentstructure via an alkyl residue. Examples include furanylmethyl,pyridinylmethyl, pyrimidinylethyl and the like.

The term “aryloxy” refers to the group —O-aryl. Similarly, “aralkoxy”and “heteroaralkoxy” refer, respectively, to an aryl or heteroarylmoiety attached to the parent structure via an alkoxy residue.

“Halogen” or “halo” refers to fluorine, chlorine, bromine or iodine(particularly fluorine, chlorine and bromine). Dihaloaryl, dihaloalkyl,trihaloaryl etc. refer to aryl and alkyl substituted with a plurality ofhalogens, but not necessarily a plurality of the same halogen; thus4-chloro-3-fluorophenyl is within the scope of dihaloaryl.

“Heterocycle” or “heterocyclyl” means a cycloalkyl or aryl residue inwhich one to four of the carbons is replaced by a heteroatom such asoxygen, nitrogen or sulfur (i.e., encompassing heterocycloalkyl andheteroaryl). Examples of heterocyclyl residues that fall within thescope of the invention include imidazolyl, imidazolinyl, pyrrolidinyl,pyrazolyl, pyrrolyl, indolyl, quinolinyl, isoquinolinyl,tetrahydroisoquinolinyl, benzofuranyl, benzodioxanyl, benzodioxolyl(commonly referred to as methylenedioxyphenyl, when occurring as asubstituent), tetrazolyl, morpholinyl, thiazolyl, pyridinyl,pyridazinyl, pyrimidinyl, thiophenyl, furanyl, oxazolyl, oxazolinyl,isoxazolyl, dioxanyl, tetrahydrofuranyl and the like. “N-heterocyclyl”refers to a nitrogen-containing heterocycle as a substituent residue.Examples of N-heterocyclyl residues include 4-morpholinyl,4-thiomorpholinyl, 1-piperidinyl, 1-pyrrolidinyl, 3-thiazolidinyl,piperazinyl and 4-(3,4-dihydrobenzoxazinyl). Examples of substitutedheterocyclyl include 4-methyl-1-piperazinyl and 4-benzyl-1-piperidinyl.

The terms “heteroaryloxy” and “heterocyclooxy” refer, respectively tothe groups —O-heteroaryl and —-O-heterocyclyl.

The term “solvate” refers to a compound (e.g., a compound of Formula Ior II or a pharmaceutically acceptable salt thereof) in physicalassociation with one or more molecules of a pharmaceutically acceptablesolvent. It will be understood that phrases such as “a compound ofFormula I or II or a pharmaceutically acceptable salt or solvatethereof” are intended to encompass the compound of Formula I or II, apharmaceutically acceptable salt of the compound, a solvate of thecompound, and a solvate of a pharmaceutically acceptable salt of thecompound.

The term “substituted” as used with regard to alkyl, aryl, aralkyl,heteroaryl and heterocyclyl refers to an alkyl, aryl, aralkyl,heteroaryl or heterocyclyl moiety wherein one or more (up to about 5,particularly up to about 3) hydrogen atoms are replaced by a substituentindependently selected from the group: optionally substituted acyl(e.g., aminocarbonyl and alkoxycarbonyl or “esters”), optionallysubstituted acyloxy (e.g., acid esters, carbamic acid esters, carbonicacid esters, and thiocarbonic acid esters), optionally substituted alkyl(e.g., fluoroalkyl), optionally substituted alkoxy (e.g., methoxy andmethoxymethoxy), alkylenedioxy (e.g. methylenedioxy), optionallysubstituted amino (e.g., alkylamino, dialkylamino, carbonylamino,benzyloxycarbonylamino or “CBZ-amino”, and carboxamido), optionallysubstituted amidino, optionally substituted aryl (e.g., phenyl and4-methyl-phenyl or “tolyl”), optionally substituted aralkyl (e.g.,benzyl), optionally substituted aryloxy (e.g., phenoxy), optionallysubstituted aralkoxy (e.g., benzyloxy), optionally substitutedheteroaryl, optionally substituted heteroaralkyl, optionally substitutedheteroaryloxy, optionally substituted heteroaralkoxy, carboxy (—COOH),cyano, halogen, hydroxy, nitro, sulfanyl, sulfinyl, sulfonyl and thio.In the compounds of Formula II where T is substituted alkylene, the term“substituted” also refers to alkylene groups where one or more (up toabout 3, particularly 1) carbon atoms are replaced by a heteroatomindependently selected from O, N or S, such as —CH₂—S—CH₂—.

The term “sulfanyl” refers to the groups: —S-(optionally substitutedalkyl), —S-(optionally substituted aryl), —S-(optionally substitutedheteroaryl), and —S-(optionally substituted heterocyclyl).

The term “sulfinyl” refers to the groups: —S(O)—H, —S(O)-(optionallysubstituted alkyl), —S(O)-(optionally substituted amino),—S(O)-(optionally substituted aryl), —S(O)-(optionally substitutedheteroaryl), and —S(O)-(optionally substituted heterocyclyl).

The term “sulfonyl” refers to the groups: —S(O)—H, —S(O₂)-(optionallysubstituted alkyl), —S(O₂)-(optionally substituted amino),—S(O₂)-(optionally substituted aryl), —S(O₂)-(optionally substitutedheteroaryl), —S(O₂)-(optionally substituted heterocyclyl),—S(O₂)-(optionally substituted alkoxy), —S(O₂)-optionally substitutedaryloxy), —S(O₂)-(optionally substituted heteroaryloxy), and—S(O₂)-(optionally substituted heterocyclyloxy).

“Isomers” are different compounds that have the same molecular formula.“Stereoisomers” are isomers that differ only in the way the atoms arearranged in space. “Enantiomers” are a pair of stereoisomers that arenon-superimposable mirror images of each other. A 1:1 mixture of a pairof enantiomers is a “racemic” mixture. The term “(.±.)” is used todesignate a racemic mixture where appropriate. “Diastereoisomers” arestereoisomers that have at least two asymmetric atoms, but which are notmirror-images of each other. The absolute stereochemistry is specifiedaccording to the Cahn-Ingold-Prelog R-S system. When a compound is apure enantiomer the stereochemistry at each chiral carbon may bespecified by either R or S. Resolved compounds whose absoluteconfiguration is unknown are designated (+) or (−) depending on thedirection (dextro- or levorotatory) which they rotate plane polarizedlight at the wavelength of the sodium D line. The term “substantiallypure” means having at least about 95% chemical purity with no singleimpurity greater than about 1%. The term “substantially optically pure”or “substantially enantiomerically pure” means having at least about 95%enantiomeric excess. The invention contemplates the use of pureenantiomers and mixtures of enantiomers, including racemic mixtures,although the use of a substantially optically pure enantiomer willgenerally be most suitable.

“Mitotic spindle formation” refers to the organization of microtubulesinto bipolar structures by mitotic kinesins. “Mitotic spindledysfunction” refers to mitotic arrest, monopolar spindle formation ormitotic spindle malformation, in which context “malformation”encompasses the splaying of mitotic spindle poles, or otherwise causingmorphological perturbation of the mitotic spindle. The term “inhibit” asused with reference to mitotic spindle formation, means altering mitoticspindle formation, including decreasing spindle formation, andincreasing or decreasing spindle pole separation. “Anti-mitotic” meansinhibiting or having the potential to inhibit mitosis, for example, asdescribed above.

The term “pharmaceutically acceptable salts” is meant to include bothacid and base addition salts. A “pharmaceutically acceptable acidaddition salt” refers to those salts that retain the biologicaleffectiveness of the free bases and that are not biologically orotherwise undesirable, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid andthe like, or organic acids such as acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like. “Pharmaceuticallyacceptable base addition salts” include those derived from inorganicbases such as sodium, potassium, lithium, ammonium, calcium, magnesium,iron, zinc, copper, manganese, aluminum salts and the like. Particularlysuitable are the ammonium, potassium, sodium, calcium, and magnesiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine.

The term “therapeutically effective amount” or “effective amount” refersto that amount of a compound of Formula I or II that is sufficient toeffect treatment, as defined below, when administered to a patient inneed of such treatment. The effective amount will vary depending uponthe patient and disease condition being treated, the weight and age ofthe patient, the severity of the disease condition, the particularcompound, pharmaceutically acceptable salt or solvate of Formula I or IIchosen, the dosing regimen to be followed, timing of administration, themanner of administration and the like, all of which can readily bedetermined by one of ordinary skill in the art. In a particular aspectof the invention, the effective amount will be an amount sufficient toinhibit KSP kinesin activity in cells involved with the disease beingtreated.

The term “treatment” or “treating” means any treatment of a disease in apatient, including:

-   -   a) preventing the disease, that is, causing the clinical        symptoms of the disease not to develop;    -   b) inhibiting the disease, that is, slowing or arresting the        development of clinical symptoms; and/or    -   c) relieving the disease, that is, causing the regression of        clinical symptoms.

A “patient” for the purposes of the present invention includes humansand other animals, particularly mammals, and other organisms. Thus themethods are applicable to both human therapy and veterinaryapplications. In a particular embodiment the patient is a mammal, mostparticularly the patient is human.

COMPOUNDS OF THE PRESENT INVENTION

The present invention provides certain quinazolinone derivatives. Thecompounds are inhibitors of one or more mitotic kinesins. The presentinvention capitalizes on the finding that perturbation of mitotickinesin function causes malformation or dysfunction of mitotic spindles,frequently resulting in cell cycle arrest and cell death.

Accordingly, the present invention relates to one or more compoundsselected from the group represented by Formula I:

where:

-   -   V is chosen from a covalent bond, CR′R″ and NR′″,        -   R′ and R″ being independently chosen from hydrogen, hydroxy,            amino, optionally substituted aryl, optionally substituted            alkylamino, optionally substituted alkyl and optionally            substituted alkoxy, and        -   R′″ being chosen from hydrogen, optionally substituted            alkyl, optionally substituted aryl, optionally substituted            aralkyl, optionally substituted heteroaryl, and optionally            substituted heteroaralkyl;    -   R¹, R², R³ and R⁴ are independently chosen from hydrogen,        hydroxy, optionally substituted alkyl, optionally substituted        alkoxy, halogen and cyano;    -   R⁵ is chosen from hydrogen, optionally substituted alkyl,        optionally substituted aryl, optionally substituted aralkyl,        optionally substituted heteroaryl, and optionally substituted        heteroaralkyl;    -   R⁶ to R⁹ are independently chosen from hydrogen, hydroxy,        optionally substituted alkyl, optionally substituted alkoxy,        optionally substituted aryl, and optionally substituted        alkylamino, provided that neither R⁸ nor R⁹ is hydroxy or alkoxy        when V is NR′″; and    -   R¹⁰ is chosen from hydrogen, optionally substituted alkyl,        optionally substituted aryl, optionally substituted aralkyl,        optionally substituted heteroaryl, and optionally substituted        heteroaralkyl,        and the pharmaceutically acceptable salts and solvates thereof.

In another aspect, the invention relates to one or more compoundsselected from the group represented by Formula II:

where:

-   -   R¹ to R¹⁰ and V are as defined with regard to Formula I;    -   T is a covalent bond or optionally substituted lower alkylene;    -   W, X, Y and Z are independently N, C, O, S or absent, provided        that:        -   no more than one of W, X, Y or Z is absent,        -   no more than two of W, X, Y and Z are —N═, and        -   W, X, Y or Z can be O or S only when one of W, X, Y or Z is            absent; and    -   R¹ to R¹⁰ and V are as defined with regard to Formula I,        provided that R¹ , R², R³ or R⁴ is absent where W, X, Y or Z,        respectively, is —N═, O, S or is absent,        including single stereoisomers, mixtures of stereoisomers, and        the pharmaceutically acceptable salts, solvates, and solvates of        pharmaceutically acceptable salts thereof. The compounds        encompassed by Formula II will be seen to include those of        Formula I; they are likewise useful as active agents in the        practice of the methods of treatment and in manufacture of        compositions including the pharmaceutical formulations of the        invention, and may also be useful as intermediates in the        synthesis of such active agents. For the sake of simplicity in        the following description and claims, substituents T, W, X, Y        and Z will not be discussed in connection with certain compounds        falling within the scope of Formula I.

Many of the compounds described herein contain one or more asymmetriccenters and may thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)— or (S)—. When the compounds described hereincontain olefinic double bonds or other centers of geometric asymmetry,and unless specified otherwise, it is intended that such compoundsinclude both E and Z geometric isomers. All tautomeric forms are alsointended to be included. The present invention is meant to include allsuch possible isomers, including racemic mixtures, intermediatemixtures, optically pure forms, substantially optically pure forms,enantiomerically pure forms, and substantially enantiomerically pureforms.

Nomenclature

The compounds of Formula I and II can be named and numbered (e.g., usingAutoNom version 2.1 in ISIS-DRAW or ChemDraw) as described below.

For example, the compound of Formula IA:

i.e., the compound according to Formula I where R¹, R² and R⁴ are H; R³is chloro; R⁵ is benzyl; R⁶ to R⁹ are H; R¹⁰ is 4-methyl-benzyl; and Vis NH, can be named3-benzyl-7-chloro-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3H-quinazolin-4-one.

The compound of Formula IB:

i.e., the compound according to Formula I where R¹, R² and R⁴ are H; R³is chloro; R⁵ is benzyl; R⁶ to R⁹ are H; R¹⁰ is benzyl; and V is CH₂,can be named3-benzyl-2-(1-benzyl-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one.

The compound of Formula IIA:

i.e., the compound according to Formula II where R¹, R² and R⁴ are H; R³is chloro; R⁵ is benzyl; R⁶ to R⁹ are H; R¹⁰ is 4-methyl-benzyl; T isisopropyl-methylene; V is NH; and W, X, Y and Z are —C═, can be named3-benzyl-7-chloro-2-{2-methyl-1-[3-(4-methyl-benzyl)-2-oxo-hexahydro-piperidin-4-yl]-propyl}-3H-quinazolin-4-one.

The compound of Formula IIB:

i.e., the compound according to Formula II where R¹, R³ and R⁴ are H; R²is di-methyl; R⁵ is benzyl; R⁶ to R⁹ are H; R¹⁰is 4-methyl-benzyl; T ismethylene; V is NR′″ where R′″ is isopropyl; and W is CH₂, X is C, and Yand Z are —C═, can be named3-benzyl-2-[1-isopropyl-3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-ylmethyl]-6,6-dimethyl-5,6-dihydro-3H-quinazolin-4-one.

The compound of Formula IIC:

i.e., the compound according to Formula II where R¹, R² and R⁴ are H; R³is chloro; R⁵ is benzyl; R⁶ to R⁹ are H; R¹⁰ is 4-methyl-benzyl; T isethylene; V is CH₂; W is —N═, and X, Y and Z are —C═, can be named3-benzyl-2-[2-(1-benzyl-6-oxo-piperidin-2-yl)-ethyl]-7-chloro-3H-pyrido[3,2-d]pyrimidin-4-one.

The compound of Formula IID:

i.e., the compound according to Formula II where R¹ and R³ are H; R² andR⁴ are absent; R⁵ is benzyl; R⁶ to R⁹ are H; R¹⁰ is benzyl; T isoxo-ethylene; V is NH; and W and Y are —C═; and X and Z are —N═, can benamed3-benzyl-2[2-(1-benzyl-6-oxo-piperidin-2-yl)-2-oxo-ethyl]-3H-pyrimidino[4,5-d]pyrimidin-4-one.

The compound of Formula IIE:

i.e., the compound according to Formula II where R¹ and R³ are H; R² andR⁴ are absent; R⁵ is benzyl; R⁶ to R¹⁰ are H; T is a covalent bond; V isNR′″ where R′″ is isopropyl; and W and Y are C; X is O, and Z is absent,can be named3-benzyl-2-(1-isopropyl-2-oxo-hexahydro-pyrimidin-4-yl)-5,7-dihydro-3H-furo[3,4-d]pyrimidin-4-one.Synthetic Reaction Parameters

The terms “solvent”, “inert organic solvent” or “inert solvent” mean asolvent inert under the conditions of the reaction being described inconjunction therewith [including, for example, benzene, toluene,acetonitrile, tetrahydrofuran (“THF”), dimethylformamide (“DMF”),chloroform, methylene chloride (or dichloromethane), diethyl ether,methanol, pyridine and the like]. Unless specified to the contrary, thesolvents used in the reactions of the present invention are inertorganic solvents.

The term “q.s.” means adding a quantity sufficient to achieve a statedfunction, e.g., to bring a solution to the desired volume (i.e., 100%).

Isolation and purification of the compounds and intermediates describedherein can be effected, if desired, by any suitable separation orpurification procedure such as, for example, filtration, extraction,crystallization, column chromatography, thin-layer chromatography orthick-layer chromatography, or a combination of these procedures.Specific illustrations of suitable separation and isolation procedurescan be had by reference to the examples hereinbelow. However, otherequivalent separation or isolation procedures can, of course, also beused.

When desired, the (R)- and (S)-isomers may be resolved by methods knownto those skilled in the art, for example by formation ofdiastereoisomeric salts or complexes which may be separated, forexample, by crystallisation; via formation of diastereoisomericderivatives which may be separated, for example, by crystallisation,gas-liquid or liquid chromatography; selective reaction of oneenantiomer with an enantiomer-specific reagent, for example enzymaticoxidation or reduction, followed by separation of the modified andunmodified enantiomers; or gas-liquid or liquid chromatography in achiral environment, for example on a chiral support, such as silica witha bound chiral ligand or in the presence of a chiral solvent. Forexample, a compound of Formula I or II can be dissolved in a loweralkanol and placed on a Chiralpak AD (205×20 mm) column (ChiralTechnologies, Inc.) conditioned for 60 min at 70% EtOAc in Hexane. Itwill be appreciated that where the desired enantiomer is converted intoanother chemical entity by one of the separation procedures describedabove, a further step may be required to liberate the desiredenantiomeric form. Alternatively, specific enantiomer may be synthesizedby asymmetric synthesis using optically active reagents, substrates,catalysts or solvents, or by converting one enantiomer to the other byasymmetric transformation.

Synthesis of the Compounds of Formula I and II

Syntheses of the compounds of Formula I and II are described below withreference to Reaction Schemes 1 to 4.

Brief Description Of Reaction Schemes

Reaction Scheme 1 illustrates a synthesis of the N,N-optionally mono- ordi-substituted pyrimidin-2-one compounds of Formula I or II.

Reaction Scheme 2 illustrates a synthesis of intermediates inpreparation of the N-optionally substituted piperidin-2-one compounds ofFormula I or II.

Reaction Scheme 3 illustrates a synthesis of intermediates inpreparation of the N-optionally mono-substituted pyrimidin-2-onecompounds of Formula I or II (i.e., where the optional substituent is atR¹⁰ and V is NH).

Reaction Scheme 4 illustrates a synthesis of the compounds of Formula Ior II from the intermediates prepared according to Reaction Schemes 2and 3.

It will be appreciated by those skilled in the art that one or more ofthe reaction steps and/or conditions described with reference toReaction Schemes 1 to 4 may require adjustment to accommodatenon-hydrogen substituents at R′, R″ and R¹ to R⁹.

Starting Materials

The N-protected 2,4-di-amino butyric acids of Formula 101 (e.g.,4-tert-butoxycarbonylamino-2-(9H-fluoren-9-yl-methoxycarbonylamino)-butyricacid), the anthranilic acids of Formula 102 (e.g., 4-chloro-anthranilicacid), aminoadipic acid hydrate, di-aminobutyric acid and the like arecommercially available, e.g., from Aldrich Chemical Company, Milwaukee,Wis. Other reactants are likewise commercially available or may bereadily prepared by those skilled in the art using commonly employedsynthetic methodology.

Preparation of Formula 103 Referring to Reaction Scheme 1, Step 1, to anN-protected di-amino lower alkyl acid of Formula 101 (e.g., a protected2,4-di-amino butyric acid, particularly employing orthogonalamino-protecting groups “PG,” such as Fmoc and Boc, to facilitateselective de-protection) in solution with an organic solvent (such asanhydrous THF) is added a slight molar excess of N-methylmorpholine, atreduced temperature (e.g., in an ice bath), followed by the addition ofa slight molar excess of isobutyl chloroformate, dropwise over 15minutes. The mixture is stirred at reduced temperature (e.g., 0° C.) foran hour, followed by the addition of a molar equivalent of an optionallysubstituted o-amino allocyclic, heterocyclic or (hetero)aryl acid ofFormula 102 (such as anthranilic acid) with continued stirring foranother 2 hours at reduced temperature. The resulting protectedalkylamino-optionally substituted cyclic acid of Formula 103 can becarried forward without isolation or purification.

Preparation of Formula 106 Referring to Reaction Scheme 1, Step 2, tothe intermediate of Formula 103 is added a slight molar excess ofN-methylmorpholine, and the mixture is allowed to warm to roomtemperature with continued stirring over 16 hours. The mixture is thencooled to 0° C. and treated with a slight molar excess of bothN-methylmorpholine and isobutyl chloroformate, followed by the roomtemperature addition of about 1.5 molar equivalents of a primary amineof formula R⁵NH₂ (such as benzylamine) in several equal portions.Removal of the solvents, partitioning between DCM and saturated sodiumbicarbonate and drying of the organic layer affords a mixture of Formula104 and Formula 105, which (in Step 3) is subsequently dried and treatedwith a molar equivalent of lithium hydroxide monohydrate in a solvent(e.g., 2/1 1,4-dioxane/ethylene glycol) at reflux for 5 hours. Thereaction is quenched with water and the desired substituted bi-cyclicproduct of Formula 106 (e.g., a quinazolinone) is extracted withdichloromethane, dried, and purified (e.g., by flash silica gelchromatography).

Preparation of Formula 107 Referring to Reaction Scheme 1, Step 4, to asolution of a compound of Formula 106 (e.g., in dichloromethane) isadded a molar equivalent of a substituted aldehyde (e.g., p-tolualdehydeor benzaldehyde). The mixture is stirred for 1 hour after which a molarexcess of sodium triacetoxyborohydride is added with continued stirringfor an additional 3 hours. The corresponding 3-substituted aminocompound of Formula 107 is conventionally isolated and purified.

Preparation of Formula 108 Referring to Reaction Scheme 1, Step 5, acompound of Formula 107 is deprotected (e.g., in the case where theprotecting group PG is t-BOC, by dissolution in aqueous TFA) and stirredfor 30 minutes, followed by evaporation of the solvents, partitioning ofthe residue and drying of the organic layer. The dried residue, andmolar excesses of DIEA, sodium triacetoxybyrohydride and an R′″-acylcompound (e.g., t-butyl N-(2-oxoethyl) carbamate) are mixed in a solvent(e.g., DCM) and stirred for 1 hour. The solution is washed, dried andevaporated to give the corresponding N—R¹⁰,N—R′-disubstituted compoundof Formula 108. As will be appreciated by those skilled in the art, thisstep can be omitted in the synthesis of compounds of Formula I or IIwhere R′″ is hydrogen, proceeding directly to either of Step 6 with thecompound of Formula 106 or 107.

Preparation of Formula I or II Referring to Reaction Scheme 1, Step 6,to a solution of Formula 106, 107 or 108 (e.g., in dichloromethane) isadded a molar excess of carbonyldiimidazole, and the reaction is stirredfor 1 hour. Evaporation of the solvent and purification (e.g., by silicagel chromatography) gives the corresponding compound of Formula I or II.Where the starting material of Formula 108 is protected (e.g., where R′is NHBoc-ethylene) the protecting group is removed (e.g., dissolving thecrude compound of Formula 1 in 95/5 TFA/H₂O with stirring for 1 hour)and the corresponding compound of Formula I or II is conventionallyisolated and purified.

Preparation of Formula 203 In Reaction Scheme 2, Step 1, to a solutionof an optionally substituted amino-dicarboxylic acid of Formula 201(e.g., aminoadipic acid hydrate dissolved in 2 molar equivalents of 2 MNaOH) is added a molar equivalent of a solution of an aldehyde ofFormula 202 (e.g., dissolved in ethanol). After 10 minutes the mixtureis cooled (e.g., to 0° C.) and sodium borohydride (0.3 molarequivalents) is added. Completion of the reaction is monitored, e.g., byLCMS, followed by extraction and isolation of a crude precipitateproduct, which is dissolved in ethanol and boiled for 16 hours to affordthe corresponding lactam intermediate of Formula 203, which can becarried forward without further purification. By substituting thecompound of Formula 201 with an optionally substitued2-amino-pentanedioic acid, the corresponding intermediates of Formula203 are obtained for the synthesis of Formula I or II where V is acovalent bond.

Preparation of Formula 204 In Reaction Scheme 2, Step 2, the lactam ofFormula 203 and a molar equivalent of DIEA are dissolved (e.g., indichloromethane) and cooled (e.g., to 0° C.). One molar equivalent ofisobutyl chloroformate is added and the mixture stirred (e.g., for 20minutes) followed by addition of 2 additional molar equivalents of DIEAfollowed by a slight molar excess of an optionally substitutedo-amino-acid of Formula 102. The reaction takes place, warming to roomtemperature, over 16 hours, to afford the corresponding acid of Formula204 (also a compound of Formula 401), which is washed, dried,evaporated, re-washed, cooled (e.g., to 0° C.), acidified and thenisolated for subsequent use without further purification.

Preparation of Formula 302 In Reaction Scheme 3, Step 1, an optionallysubstituted diamino lower alkyl acid of Formula 301 and 3 molarequivalents of sodium bicarbonate are dissolved in water, to whichone-half molar equivalent of copper sulfate dissolved in water is added.A molar excess of di-(tert-butyl) pyrocarbonate (dissolved, e.g., inacetone) is added followed by stirring for 24 hours, the addition ofmethanol and continued stirring for another 18 hours. The resultingintermediate mono-Boc-protected copper complex is filtered, washed anddried, then suspended in water. Two molar equivalents of quinolol areadded to the suspension. After 5 hours, the suspension is filtered offand the liquid is evaporated. The solid thus-obtained is dissolved(e.g., in 200 mL of 30% methanol in benzene), and(trimethylsilyl)diazomethane is added, dropwise to completion (indicatedby color change and cessation of bubbling), followed by stirring for 1hour and the dropwise addition of acetic acid to completion (indicated,e.g., by color change and cessation of bubbling). The resulting materialis purified conventionally to provide the corresponding methyl ester ofFormula 302.

Preparation of Formula 303 In Reaction Scheme 3, Step 2, to a solutionof Formula 302 (e.g., in DCM) is added almost one molar equivalent of anR¹⁰-aldehyde (e.g., p-tolualdehyde or benzaldehyde) and the mixturestirred at room temperature for 1 hour. A slight molar excess of sodiumtriacetoxyborohydride is added and the mixture is stirred for 16 hours.The protecting group is removed (e.g., dissolving the R¹⁰-substitutedamine in 2M HCl in dioxane solution followed by stirring for 2 hours).The solution is then washed, dried, isolated and purified conventionallyto give the corresponding optionally substituted-aminomethyl estercompound of Formula 303.

Preparation of Formula 304 In Reaction Scheme 3, Step 3, to a solutionof Formula 303 and 2 molar equivalents of DIEA (e.g., in DCM) is added amolar excess of carbonyldiimidazole. The reaction mixture is stirred for1 hour, after which the solvents are evaporated. The residue isdissolved (e.g., in MeOH:H₂O (2:1) solution) to which solution is wasadded 2 molar equivalents of LiOH. The reaction takes place withstirring over 3 hours after which pH is adjusted to ˜7 (e.g., by addingDowex-H+ resin). Conventional isolation and purification gives thecorresponding pyrimidine of Formula 304.

Preparation of Formula 305 In Reaction Scheme 3, Step 4, to a solutionof Formula 304 (e.g., in DMF) is added a slight molar excess ofanhydrous N-methylmorpholine. After cooling in an ice-bath for 10minutes, a slight molar excess of isobutyl chloroformate is addeddropwise while maintaining the temperature below 5° C. with stirring for1 hour. A slight molar excess of an optionally substituted o-aminoallocyclic, heterocyclic or (hetero)aryl acid of Formula 102 (such asanthranilic acid) of Formula 102 (dissolved, e.g., in DMF) is added andthe mixture stirred for an additional 5 hours during which thetemperature is allowed to warm to room temperature to afford thecorresponding intermediate product of Formula 305 (also a compound ofFormula 401), which can be carried on without isolation or purification.

Preparation of Formula 402 and Formula I or II In Reaction Scheme 4,Step 1, to a solution of a piperidine compound of Formula 401 (e.g., inDMF) is added 3 molar equivalents of EDC followed by stirring at roomtemperature for 1 hour. Three molar equivalents of an R⁵-amine (e.g.,benzylamine) are added followed by stirring for an additional 3 hours.Conventional isolation and purification provides the corresponding crudeintermediate of Formula 402. In Reaction Scheme 4, Step 2, the compoundof Formula 402 is added to a mixture of ethylene glycol with 1 molarequivalent of sodium hydroxide, followed by stirring at 130° C. for twodays. The mixture is then poured into water, extracted and purified togive the corresponding pure product of Formula I or II.

Alternative Preparation of Formula 402 and Formula I or IIAlternatively, in Reaction Scheme 4, Step 1, two molar equivalents ofEDC are added to a solution of a pyrimidine compound of Formula 401(e.g., in DMF) followed by stirring for 1 hour and then by the additionof an R⁵-amine (e.g., benzylamine). The resulting solution is stirredfor 16 hours and the corresponding compound of Formula 402 is isolatedand purified conventionally. In Reaction Scheme 4, Step 2, the compoundof Formula 402 is dissolved (e.g., in ethylene glycol to which was added2 molar equivalents of sodium hydroxide). The mixture is stirred at 140°C. for 20 hours. Following consumption of starting material, thereaction mixture is poured into 100 mL of water. After extraction withDCM, the crude product is isolated and purified conventionally to givethe corresponding pure product of Formula I or II.

Compounds prepared by the above-described process of the invention maybe identified by the presence of a detectable amount of Formula 402.While it is well known that pharmaceuticals must meet pharmacopoeiastandards before approval and/or marketing, and that synthetic reagents(such as benzylamine, ethylene glycol or NaOH) and precursors (such asFormula 402) should not exceed the limits prescribed by pharmacopoeiastandards, final compounds prepared by a process of the presentinvention may have minor, but detectable, amounts of such materialspresent, for example at levels in the range of 95% purity with no singleimpurity greater than 1%. These levels can be detected, e.g., byemission spectroscopy. It is important to monitor the purity ofpharmaceutical compounds for the presence of such materials, whichpresence is additionally disclosed as a method of detecting use of aprocess of the invention.

Particular Optional Processes and Last Steps

A compound of Formula 402 is dissolved in an organic solvent (e.g.,ethylene glycol) to which was added about 2 molar equivalents of sodiumhydroxide.

A protected amino-substituted precursor to Formula I or II (e.g. whereR′″ or R¹⁰ is NHBoc-protected amino ethyl) is dissolved in TFA/H₂O andstirred to afford the corresponding de-protected compound of Formula Ior II.

A racemic mixture of isomers of a compound of Formula I or II is placedon a chromatography column and separated into (R)- and (S)-enantiomers.

A compound of Formula I or II is contacted with a pharmaceuticallyacceptable acid to form the corresponding acid addition salt.

A pharmaceutically acceptable acid addition salt of Formula I or II iscontacted with a base to form the corresponding free base of Formula Ior II.

Particular Compounds

Particular embodiments of the invention include or employ the compoundsof Formula I and II having the following combinations and permutationsof substituent groups (indented/sub-grouped, respectively, in increasingorder of particularity). These are presented in support of the appendedclaims as well as combinations and permutations of substituent groupsthat may, for the sake of brevity, not be specifically claimed butshould be appreciated as encompassed by the teachings of the presentdisclosure. In that regard, the described subsets for each substituentare intended to apply to that substituent alone or in combination withone, several, or all of the described subsets for the othersubstituents, for example, as illustrated with regard to the compoundswhere V is CR′R″ or NR′″.

-   W, X, Y and Z are independently chosen from —C═ and —N═;    -   W, X, Y and Z are —C═.-   R¹, R², R³ and R⁴ are independently chosen from hydrogen, halo    (especially chloro and fluoro), lower alkyl (especially methyl),    substituted lower alkyl, lower alkoxy (especially methoxy), and    cyano.    -   R¹, R², R³ and R⁴ are independently hydrogen, chloro, fluoro,        methyl, methoxy or cyano.    -   Where three or four of R¹, R², R³ and R⁴ are hydrogen.        -   Where four of R¹, R², R³ and R⁴ are hydrogen or three of R¹,            R², R³ and R⁴ are hydrogen and the fourth is halo, methoxy,            methyl, or cyano.            -   Where halo is chloro.                -   Where R³ is hydrogen or chloro.                -    Where R³ is chloro.-   R⁵ is optionally substituted aralkyl.    -   R⁵ is benzyl or substituted benzyl.        -   R⁵ is benzyl.-   R⁶ is hydrogen or optionally substituted lower alkyl.    -   R⁶ is hydrogen.-   R⁷ is hydrogen or optionally substituted lower alkyl.    -   R⁷ is hydrogen.-   R⁸ is hydrogen or optionally substituted lower alkyl.    -   R⁸ is hydrogen.-   R⁹ is hydrogen or optionally substituted lower alkyl.    -   R⁹ is hydrogen.-   R¹⁰ is optionally substituted aryl or optionally substituted    aralkyl.    -   R¹⁰ is optionally substituted phenyl or optionally substituted        benzyl.        -   R¹⁰ is benzyl or methyl-benzyl.-   T is optionally substituted C₁ to C₄ alkylene or is absent.    -   T is absent.    -   Where T is alkylene having a carbon substituted by a heteroatom,        the heteroatom is not bound directly to the bicyclic structure.        -   T is aminoalkylene or amidoalkylene.    -   T is alkylene or alkylene substituted with halo or oxo.-   V is CR′R″ or NR′″    -   V is CR′R″ (particularly where R′ and/or R″ are hydrogen).        -   Where R′ and R″ are hydrogen.        -   Where W, X, Y and Z are independently chosen from —C═ and            —N═.            -   W, X, Y and Z are —C═.        -   Where R¹, R², R³ and R⁴ are independently chosen from            hydrogen, halo (particularly chloro and fluoro), lower alkyl            (particularly methyl), substituted lower alkyl, lower alkoxy            (particularly methoxy), and cyano.            -   R¹, R², R³ and R⁴ are independently hydrogen, chloro,                fluoro, methyl, methoxy or cyano.            -   Where three or four of R¹, R², R³ and R⁴ are hydrogen.                -   Where four of R¹, R², R³ and R⁴ are hydrogen or                    three of R¹, R², R³ and R⁴ are hydrogen and the                    fourth is halo, methoxy, methyl, or cyano.                -    Where halo is chloro.                -    Where R³ is chloro.                -    Where R′ and R″ are hydrogen.        -   Where R⁵ is optionally substituted aralkyl.            -   Where R⁵ benzyl or substituted benzyl.                -   Where R⁵ is benzyl.            -   Where R′ and R″ are hydrogen.        -   Where R⁶, R⁷, R⁸ and R⁹ are independently chosen from            hydrogen or optionally substituted lower alkyl.            -   Where at least three of R⁶, R⁷, R⁸ and R⁹ are hydrogen.                -   Where R⁶, R⁷, R⁸ and R⁹ are hydrogen.                -    Where R′ and R″ are hydrogen.        -   Where R¹⁰ is optionally substuted aryl or optionally            substituted aralkyl.            -   R¹⁰ is optionally substituted phenyl or optionally                substituted benzyl.                -   R¹⁰ is benzyl or methyl-benzyl.                -    Where R′ and R″ are hydrogen.        -   Where W, X, Y and Z are chosen from —C═ and —N═.            -   W, X, Y and Z are —C═.                -   Where R¹, R², R³ and R⁴ are independently chosen                    from hydrogen, halo (particularly chloro and                    fluoro), lower alkyl (particularly methyl),                    substituted lower alkyl, lower alkoxy (particularly                    methoxy), and cyano.                -    R¹, R², R³ and R⁴ are independently hydrogen,                    chloro, fluoro, methyl, methoxy or cyano.                -    Where three or four of R¹, R², R³ and R⁴ are                    hydrogen.                -    Where four of R¹, R², R³ and R⁴ are hydrogen or                    three of R¹, R², R³ and R⁴ are hydrogen and the                    fourth is halo, methoxy, methyl, or cyano.                -    Where halo is chloro.                -    Where R³ is chloro.        -   Where R¹, R², R³ and R⁴ are independently chosen from            hydrogen, halo (particularly chloro and fluoro), lower alkyl            (particularly methyl), substituted lower alkyl, lower alkoxy            (particularly methoxy), and cyano.            -   R¹, R², R³ and R⁴ are independently hydrogen, chloro,                fluoro, methyl, methoxy or cyano.            -   Where three or four of R¹, R², R³ and R⁴ are hydrogen.                -   Where four of R¹, R², R³ and R⁴ are hydrogen or                    three of R¹, R², R³ and R⁴ are hydrogen and the                    fourth is halo, methoxy, methyl, or cyano.                -    Where halo is chloro.                -    Where R³ is chloro.        -   Where R⁵ is optionally substituted aralkyl.            -   Where R⁵ is benzyl or substituted benzyl.                -   Where R⁵is benzyl.        -   Where R⁶, R⁷, R⁸ and R⁹ are independently chosen from            hydrogen or optionally substituted lower alkyl.            -   Where at least three of R⁶, R⁷, R⁸ and R⁹ are hydrogen.                -   Where R⁶, R⁷, R⁸ and R⁹ are hydrogen.        -   Where R¹⁰ is optionally substituted aryl or optionally            substituted aralkyl.            -   R¹⁰ is optionally substituted phenyl or optionally                substituted benzyl.                -   R¹⁰ is benzyl or methyl-benzyl.        -   Where T is optionally substituted C₁ to C₄ alkylene or is            absent.            -   T is absent.            -   Where T is alkylene having a carbon substituted by a                heteroatom, the heteroatom is not bound directly to the                bicyclic structure.                -   T is aminoalkylene or amidoalkylene.            -   T is alkylene or alkylene substituted with halo or oxo.    -   V is NR′″ (particularly where R′″ is hydrogen or optionally        substituted alkyl).        -   Where R″ is hydrogen or optionally substituted amino-lower            alkyl.        -   Where W, X, Y and Z are chosen from —C═ and —N═.            -   W, X, Y and Z are —C═.        -   Where R¹, R², R³ and R⁴ are independently chosen from            hydrogen, halo (particularly chloro and fluoro), lower alkyl            (particularly methyl), substituted lower alkyl, lower alkoxy            (particularly methoxy), and cyano.            -   R¹, R², R³ and R⁴ are independently hydrogen, chloro,                fluoro, methyl, methoxy or cyano.            -   Where three or four of R¹, R², R³ and R⁴ are hydrogen.                -   Where four of R¹, R², R³ and R⁴ are hydrogen or                    three of R¹, R², R³ and R⁴ are hydrogen and the                    fourth is halo, methoxy, methyl, or cyano.                -    Where halo is chloro.                -    Where R³ is chloro.                -    Where R′″ is hydrogen or amino-lower alkyl.        -   Where R⁵ is optionally substituted aralkyl.            -   Where R⁵is benzyl or substituted benzyl.                -   Where R⁵ is benzyl.            -   Where R′″ is hydrogen or optionally substituted                amino-lower alkyl.        -   Where R⁶, R⁷, R⁸ and R⁹ are independently chosen from            hydrogen or optionally substituted lower alkyl.            -   Where at least three of R⁶, R⁷, R⁸ and R⁹ are hydrogen.                -   Where R⁶, R⁷, R⁸ and R⁹ are hydrogen.                -    Where R′″ is hydrogen or amino-lower alkyl.        -   Where R¹⁰ is optionally substuted aryl or optionally            substituted aralkyl.            -   R¹⁰ is optionally substituted phenyl or optionally                substituted benzyl.                -   R¹⁰ is benzyl or methyl-benzyl.                -    Where R′″ is hydrogen or amino-lower alkyl.        -   Where W, X, Y and Z are chosen from —C═ and —N═.            -   W, X, Y and Z are —C═.                -   Where R¹, R², R³ and R⁴ are independently chosen                    from hydrogen, halo (particularly chloro and                    fluoro), lower alkyl (particularly methyl),                    substituted lower alkyl, lower alkoxy (particularly                    methoxy), and cyano.                -    R¹, R², R³ and R⁴ are independently hydrogen,                    chloro, fluoro, methyl, methoxy or cyano.                -    Where three or four of R¹, R², R³ and R⁴ are                    hydrogen.                -    Where four of R¹, R², R³ and R⁴ are hydrogen or                    three of R¹, R², R³ and R⁴ are hydrogen and the                    fourth is halo, methoxy, methyl, or cyano.                -    Where halo is chloro.                -    Where R³is chloro.        -   Where R¹, R², R³ and R⁴ are independently chosen from            hydrogen, halo (particularly chloro and fluoro), lower alkyl            (particularly methyl), substituted lower alkyl, lower alkoxy            (particularly methoxy), and cyano.            -   R¹, R², R³ and R⁴ are independently hydrogen, chloro,                fluoro, methyl, methoxy or cyano.            -   Where three or four of R¹, R², R³ and R⁴ are hydrogen.                -   Where four of R¹, R², R³ and R⁴ are hydrogen or                    three of R¹, R², R³ and R⁴ are hydrogen and the                    fourth is halo, methoxy, methyl, or cyano.                -    Where halo is chloro.                -    Where R³is chloro.        -   Where R⁵ is optionally substituted aralkyl.            -   Where R⁵ is benzyl or substituted benzyl.                -   Where R⁵ is benzyl.        -   Where R⁶, R⁷, R⁸ and R⁹ are independently chosen from            hydrogen or optionally substituted lower alkyl.            -   Where at least three of R⁶, R⁷, R⁸ and R⁹ are hydrogen.                -   Where R⁶, R⁷, R⁸ and R⁹ are hydrogen.        -   Where R¹⁰ is optionally substituted aryl or optionally            substituted aralkyl.            -   R¹⁰ is optionally substituted phenyl or optionally                substituted benzyl.                -   R¹⁰ is benzyl or methyl-benzyl.        -   Where T is optionally substituted C₁ to C₄ alkylene or is            absent.            -   T is absent.            -   Where T is alkylene having a carbon substituted by a                heteroatom, the heteroatom is not bound directly to the                bicyclic structure.                -   T is aminoalkylene or amidoalkylene.            -   T is alkylene or alkylene substituted with halo or oxo.

Compounds where V is CR′R″ or NR′″, including those illustrated by theabove-described groupings and sub-groups of substituents, individuallyand/or combined together, are particularly suitable for practice of thepresent invention.

One group of compounds, pharmaceutically acceptable salts and solvatesthereof, compositions including pharmaceutical formulations, and methodsof manufacture and use of the present invention are those wherein thecompound of Formula I or II is selected from:

-   -   3-benzyl-7-chloro-2-[3-benzyl-2-oxo-hexahydro-pyrimidin-4-yl]-3H-quinazolin-4-one;    -   3-benzyl-7-chloro-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3H-quinazolin-4-one;    -   3-benzyl-2-(1-benzyl-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one;    -   3-benzyl-2-(1-(4-methyl-benzyl)-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one;    -   2-[-1-(2-amino-ethyl)-3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3-benzyl-7-chloro-3H-quinazolin-4-one.

A particular group of compounds, pharmaceutically acceptable salts andsolvates thereof, compositions including pharmaceutical formulations,and methods of manufacture and use of the present invention are thosewherein the compound of Formula I or II is selected from:

-   -   3-benzyl-7-chloro-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3H-quinazolin-4-one;    -   3-benzyl-2-(1-benzyl-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one;        and    -   3-benzyl-2-(1-(4-methyl-benzyl)-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one.

Another particular group of compounds, pharmaceutically acceptable saltsand solvates thereof, compositions including pharmaceuticalformulations, and methods of manufacture and use of the presentinvention are those wherein the compound of Formula I or II is selectedfrom:

-   -   3-benzyl-2-(1-benzyl-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one;        and    -   3-benzyl-2-(1-(4-methyl-benzyl)-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one.

Utility, Testing and Administration

General Utility

The compounds of the invention find use in a variety of applications,including as therapeutic active agents, in the practice of the methodsof treatment, in compositions, particularly pharmaceutical formulationsand in methods for the manufacture of pharmaceutical formulations, andas intermediates in the synthesis of such therapeutic active agents.

As will be appreciated by those in the art, mitosis can be altered in avariety of ways; that is, one can affect mitosis either by increasing,decreasing or otherwise interfering with the activity of a component inthe mitotic pathway. Stated differently, mitosis can be affected (e.g.,disrupted) by disturbing equilibrium, either by inhibiting or activatingcertain mitotic components. Similar approaches can be used to altermeiosis.

The compounds of the invention can be used to inhibit mitotic spindleformation. Such inhibition may take the form of lessening a mitotickinesin's organization of microtubules into bipolar structures,increasing or decreasing spindle pole separation, and/or inducingmitotic spindle dysfunction. In particular, the compounds of theinvention are useful to bind to and/or inhibit the activity of a mitotickinesin, KSP, especially human KSP, although KSP kinesins from otherorganisms may also be used. Also included within the definition of theterm “KSP” for these purposes are variants and/or fragments of KSP. See,U.S. Pat. No. 6,437,115. While other mitotic kinesins may be used in thepresent invention, the compounds of the invention have been shown tohave specificity for KSP. Contacting a compound of the invention with aKSP kinesin, particularly human KSP kinesin, can lead to diminishedKSP-mediated ATP hydrolysis activity and/or diminished KSP-mediatedmitotic spindle formation activity. Meiotic spindles can be similarlydisrupted.

In another embodiment, the compounds of the invention can be used tomodulate one or more other human mitotic kinesins, in addition toinhibiting KSP, including: HSET (see, U.S. Pat. No. 6,361,993); MCAK(see, U.S. Pat. No. 6,331,424); CENP-E (see, PCT Publication No. WO99/13061); Kif4 (see, U.S. Pat. No. 6,440,684); MKLP1 (see, U.S. Pat.No. 6,448,025); Kif15 (see, U.S. Pat. No. 6,355,466); Kid (see, U.S.Pat. No. 6,387,644); Mpp1, CMKrp, KinI-3 (see, U.S. Pat. No. 6,461,855);Kip3a (see, PCT Publication No. WO 01/96593); Kip3d (see, U.S. Pat. No.6,492,151); and RabK6.

Therapeutic uses facilitated by the mitotic kinesin-inhibitory activityof the compounds of the present invention include the treatment ofdisorders associated with cell proliferation. Particular disease statesthat can be treated by the methods, pharmaceutical formulations, andcompounds provided herein include, but are not limited to, cancer(further discussed below), autoimmune disease, arthritis, graftrejection, inflammatory bowel disease, proliferation induced aftermedical procedures, including, but not limited to, surgery, angioplasty,and the like. In one embodiment, the invention includes application tocells or individuals afflicted or impending afflication with any one ofthese disorders or states.

The compounds, pharmaceutical formulations and methods provided hereinare particularly deemed useful for the treatment of cancer includingsolid tumors such as skin, breast, brain, cervical carcinomas,testicular carcinomas, etc. More particularly, cancers that can betreated include, but are not limited to:

-   -   Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma,        liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma;    -   Lung: bronchogenic carcinoma (squamous cell, undifferentiated        small cell, undifferentiated large cell, adenocarcinoma),        alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma,        lymphoma, chondromatous hamartoma, mesothelioma;    -   Gastrointestinal: esophagus (squamous cell carcinoma,        adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma,        lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma,        insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma),        small bowel (adenocarcinoma, lymphoma, carcinoid tumors,        Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma,        fibroma), large bowel (adenocarcinoma, tubular adenoma, villous        adenoma, hamartoma, leiomyoma);    -   Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor        [nephroblastoma], lymphoma, leukemia), bladder and urethra        (squamous cell carcinoma, transitional cell carcinoma,        adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis        (seminoma, teratoma, embryonal carcinoma, teratocarcinoma,        choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,        fibroadenoma, adenomatoid tumors, lipoma);    -   Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma,        hepatoblastoma, angiosarcoma, hepatocellular adenoma,        hemangioma;    -   Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant        fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant        lymphoma (reticulum cell sarcoma), multiple myeloma, malignant        giant cell tumor chordoma, osteochronfroma (osteocartilaginous        exostoses), benign chondroma, chondroblastoma,        chondromyxofibroma, osteoid osteoma and giant cell tumors;    -   Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,        osteitis deformans), meninges (meningioma, meningiosarcoma,        gliomatosis), brain (astrocytoma, medulloblastoma, glioma,        ependymoma, germinoma [pinealoma], glioblastoma multiform,        oligodendroglioma, schwannoma, retinoblastoma, congenital        tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma);    -   Gynecological: uterus (endometrial carcinoma), cervix (cervical        carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian        carcinoma [serous cystadenocarcinoma, mucinous        cystadenocarcinoma, unclassified carcinoma], granulosa-thecal        cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant        teratoma), vulva (squamous cell carcinoma, intraepithelial        carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina        (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma        (embryonal rhabdomyosarcoma], fallopian tubes (carcinoma);    -   Hematologic: blood (myeloid leukemia [acute and chronic], acute        lymphoblastic leukemia, chronic lymphocytic leukemia,        myeloproliferative diseases, multiple myeloma, myelodysplastic        syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant        lymphoma];    -   Skin: malignant melanoma, basal cell carcinoma, squamous cell        carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma,        angioma, dermatofibroma, keloids, psoriasis; and    -   Adrenal glands: neuroblastoma.        As used herein, treatment of cancer includes treatment of        cancerous cells, including cells afflicted by any one of the        above-identified conditions.

Another useful aspect of the invention is a kit having a compound, saltor solvate of Formula I or II and a package insert or other labelingincluding directions treating a cellular proliferative disease byadministering an effective amount of the compound, salt or solvate. Thecompound, salt or solvate of Formula I or II in the kits of theinvention is particularly provided as one or more doses for a course oftreatment for a cellular proliferative disease, each dose being apharmaceutical formulation including a pharmaceutically acceptedexcipient and a compound, salt or solvate of Formula I or II.

Testing

To assay activity, generally, either KSP or a compound according to theinvention is non-diffusably bound to an insoluble support havingisolated sample receiving areas. The insoluble support can be made ofany material to which the compounds can be bound, is readily separatedfrom soluble material, and is otherwise compatible with the overallmethod of screening. The surface of such supports can be solid or porousand of any convenient shape. Examples of suitable insoluble supportsinclude microtiter plates, arrays, membranes and beads. These aretypically made of glass, plastic (e.g., polystyrene), polysaccharides,nylon or nitrocellulose, Teflon™, etc. Microtiter plates and arrays areespecially convenient because a large number of assays can be carriedout simultaneously, using small amounts of reagents and samples. Theparticular manner of binding of the compound is not crucial so long asit is compatible with the reagents and overall methods of the invention,maintains the activity of the compound and is nondiffusable. Particularmethods of binding include the use of antibodies (which do notsterically block either the ligand binding site or activation sequencewhen the protein is bound to the support), direct binding to “sticky” orionic supports, chemical crosslinking, the synthesis of the protein oragent on the surface, etc. Following binding of the protein or agent,excess unbound material is removed by washing. The sample receivingareas can then be blocked through incubation with bovine serum albumin(BSA), casein or other innocuous protein or other moiety.

The compounds of the invention can be used on their own to modulate theactivity of a mitotic kinesin, particularly KSP. In this embodiment, acompound of the invention is combined with KSP and the activity of KSPis assayed. Measurable kinesin activities include the ability to affectATP hydrolysis; microtubule binding; gliding andpolymerization/depolymerization (effects on microtubule dynamics);binding to other proteins of the spindle; binding to proteins involvedin cell-cycle control; serving as a substrate to other enzymes, such askinases or proteases; and specific kinesin cellular activities such asspindle pole separation.

Methods of performing motility assays are well known to those of skillin the art. [See e.g., Hall, et al. (1996), Biophys. J., 71: 3467-3476,Turner et al., 1996, Anal. Biochem. 242 (1):20-5; Gittes et al., 1996,Biophys. J. 70(I): 418-29; Shirakawa et al., 1995, J. Exp. Biol. 198:1809-15; Winkelmann et al., 1995, Biophys. J. 68: 2444-53; Winkelmann etal., 1995, Biophys. J. 68: 72S.]

Methods known in the art for determining ATPase hydrolysis activity alsocan be used. Solution based assays are particularly suitable (see, U.S.Pat. No. 6,410,254); alternatively, conventional methods are used. Forexample, P_(i) release from kinesin can be quantified. In oneembodiment, the ATPase hydrolysis activity assay utilizes 0.3 M PCA(perchloric acid) and malachite green reagent (8.27 mM sodium molybdateII, 0.33 mM malachite green oxalate, and 0.8 mM Triton X-100). Toperform the assay, 10 μL of reaction is quenched in 90 μL of cold 0.3 MPCA. Phosphate standards are used so data can be converted to mMinorganic phosphate released. When all reactions and standards have beenquenched in PCA, 100 μL of malachite green reagent is added to therelevant wells in e.g., a microtiter plate. The mixture is developed for10-15 minutes and the plate is read at an absorbance of 650 nm. Whenphosphate standards are used, absorbance readings can be converted to mMP_(i) and plotted over time. Additionally, ATPase assays known in theart include the luciferase assay.

ATPase activity of kinesin motor domains also can be used to monitor theeffects of modulating agents. In one embodiment ATPase assays of kinesinare performed in the absence of microtubules. In another embodiment, theATPase assays are performed in the presence of microtubules. Differenttypes of modulating agents can be detected in the above assays. In oneparticular embodiment, the effect of a modulating agent is independentof the concentration of microtubules and ATP. In another embodiment, theeffect of the agents on kinesin ATPase can be decreased by increasingthe concentrations of ATP, microtubules or both. In yet anotherembodiment, the effect of the modulating agent is increased byincreasing concentrations of ATP, microtubules or both.

Agents that modulate the biochemical activity of KSP in vitro may thenbe screened in vivo. Methods for testing such agents in vivo includeassays of cell cycle distribution, cell viability, or the presence,morphology, activity, distribution or amount of mitotic spindles.Methods for monitoring cell cycle distribution of a cell population, forexample, by flow cytometry, are well known to those skilled in the art,as are methods for determining cell viability. See, for example, WO01/31335, entitled “Methods of Screening for Modulators of CellProliferation and Methods of Diagnosing Cell Proliferation States.”

In addition to the assays described above, microscopic methods formonitoring spindle formation and malformation are well known to those ofskill in the art (see, e.g., Whitehead and Rattner (1998), J. Cell Sci.111:2551-61; Galgio et al, (1996) J. Cell Biol., 135:399-414).

The compounds of the invention inhibit KSP kinesin. One measure ofinhibition, IC₅₀, is defined as the concentration of the compound atwhich the activity of KSP is decreased by fifty percent. Particularlysuitable compounds have IC₅₀'s of less than about 1 mM, with moreparticularly suitable compounds having IC₅₀'s of less than about 100 μM.IC₅₀'s of less than about 10 nM can be attained by certain compounds ofthe invention, and the pharmaceutically acceptable salts and solvatesthereof, it being appreciated that a smaller IC₅₀ is generallyconsidered advantageous. Measurement of IC₅₀ is done using an ATPaseassay.

Another measure of inhibition is K_(i). For compounds with IC₅₀'s lessthan 1 μM, the K_(i) or Kis defined as the dissociation rate constantfor the interaction of the test compound with KSP. Particularly suitablecompounds have K_(i)'s of less than about 100 μM, more particularlysuitable compounds having K_(i)'s of less than about 10 μM. K_(i)'s ofless than about 10 nM can be attained by certain compounds of theinvention, and the pharmaceutically acceptable salts and solvatesthereof, it being appreciated that a smaller K_(i) is generallyconsidered advantageous. The K_(i) for a compound is determined from theIC₅₀ based on three assumptions. First, only one compound molecule bindsto the enzyme and there is no cooperativity. Second, the concentrationsof active enzyme and the compound tested are known (i.e., there are nosignificant amounts of impurities or inactive forms in thepreparations). Third, the enzymatic rate of the enzyme-inhibitor complexis zero. The rate (i.e., compound concentration) data are fitted to theequation:$V = {V_{\max}{E_{0}\left\lbrack {I - \frac{\left( {E_{0} + I_{0} + {Kd}} \right) - \sqrt{\left( {E_{0} + I_{0} + {Kd}} \right)^{2} - {4E_{0}I_{0}}}}{2E_{0}}} \right\rbrack}}$Where V is the observed rate, V_(max) is the rate of the free enzyme, I₀is the inhibitor concentration, E₀ is the enzyme concentration, andK_(d) is the dissociation constant of the enzyme-inhibitor complex.

Another measure of inhibition is GI₅₀, defined as the concentration ofthe compound that results in a decrease in the rate of cell growth byfifty percent. Anti-proliferative compounds that have been successfullyapplied in the clinic to treatment of cancer (cancer chemotherapeutics)have GI₅₀'s that vary greatly. For example, in A549 cells, paclitaxelGI₅₀ is 4 nM, doxorubicin is 63 nM, 5-fluorouracil is 1 μM, andhydroxyurea is 500 μM (data provided by National Cancer Institute,Developmental Therapeutic Program, http://dtp.nci.nih.gov/). Therefore,compounds that inhibit cellular proliferation at virtually anyconcentration may be useful. Particularly suitable compounds have GI₅₀'sof less than about 1 mM, with more particularly suitable compoundshaving a GI₅₀ of less than about 10 μM. GI₅₀'s of less than about 10 nMcan be attained by certain compounds of the invention, and thepharmaceutically acceptable salts and solvates thereof, it beingappreciated that a smaller GI₁₅ is generally considered advantageous.Measurement of GI₅₀ is done using a cell proliferation assay.

Testing for growth inhibition using cell lines (such as MCF-7/ADR-RESand HCT15) that express P-glycoprotein (also known as Multi-drugResistance, or MDR⁺), which conveys resistance to other chemotherapeuticdrugs, such as pacilitaxel, can identify anti-mitotic agents thatinhibit cell proliferation and are not subject to resistance byoverexpression of MDR⁺ by drug-resistant tumor lines.

In vitro potency of small molecule inhibitors is determined by assayinghuman ovarian cancer cells (SKOV3) for viability following a 72-hourexposure to a 9-point dilution series of compound. Cell viability isdetermined by measuring the absorbance of formazon, a product formed bythe bioreduction of MTS/PMS, a commercially available reagent. Eachpoint on the dose-response curve is calculated as a percent of untreatedcontrol cells at 72 hours minus background absorption (complete cellkill).

To employ the compounds of the invention in a method of screening forcompounds that bind to KSP kinesin, the KSP is bound to a support, and acompound or composition of the invention is added to the assay.Alternatively, a composition of a compound of the invention bound to asolid support can be made, and KSP added to the assay. Classes ofcompounds among which novel binding agents may be sought includespecific antibodies, non-natural binding agents identified in screens ofchemical libraries, peptide analogs, etc. Of particular interest arescreening assays for candidate agents that have a low toxicity for humancells. A wide variety of assays may be used for this purpose, includinglabeled in vitro protein-protein binding assays, electrophoreticmobility shift assays, immunoassays for protein binding, functionalassays (phosphorylation assays, etc.) and the like.

The determination of the binding of the mitotic agent to KSP may be donein a number of ways. In a particular embodiment, the compound of theinvention is labeled, for example, with a fluorescent or radioactivemoiety and binding determined directly. For example, this may be done byattaching all or a portion of KSP to a solid support, adding a labeledcompound (for example a compound of the invention in which at least oneatom has been replaced by a detectable isotope), washing off excessreagent, and determining whether the amount of the label is that presenton the solid support. Various blocking and washing steps may be utilizedas is known in the art.

By “labeled” herein is meant that the compound is either directly orindirectly labeled with a label which provides a detectable signal,e.g., radioisotope, fluorescent tag, enzyme, antibodies, particles suchas magnetic particles, chemiluminescent tag, or specific bindingmolecules, etc. Specific binding molecules include pairs, such as biotinand streptavidin, digoxin and antidigoxin etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined above. The label can directly or indirectlyprovide a detectable signal.

In some embodiments, only one of the components is labeled. For example,the kinesin proteins may be labeled at tyrosine positions using ¹²⁵I, orwith fluorophores. Alternatively, more than one component may be labeledwith different labels; using ¹²⁵I for the proteins, for example, and afluorophor for the anti-mitotic agents.

The compounds of the invention may also be used as competitors to screenfor additional drug candidates. “Candidate agent” or “drug candidate” orgrammatical equivalents as used herein describe any molecule, e.g.,protein, oligopeptide, small organic molecule, polysaccharide,polynucleotide, etc., to be tested for bioactivity. They may be capableof directly or indirectly altering the cellular proliferation phenotypeor the expression of a cellular proliferation sequence, including bothnucleic acid sequences and protein sequences. In other cases, alterationof cellular proliferation protein binding and/or activity is screened.Screens of this sort may be performed either in the presence or absenceof microtubules. In the case where protein binding or activity isscreened, particular embodiments exclude molecules already known to bindto that protein, for example, polymer structures such as microtubules,and energy sources such as ATP. Particular embodiments of assays hereininclude candidate agents that do not bind the cellular proliferationprotein in its endogenous native state termed herein as “exogenous”agents. In another particular embodiment, exogenous agents furtherexclude antibodies to KSP.

Candidate agents can encompass numerous chemical classes, thoughtypically they are organic molecules, particularly small organiccompounds having a molecular weight of more than 100 and less than about2,500 daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding andlipophilic binding, and typically include at least an amine, carbonyl,hydroxyl, ether, or carboxyl group, especially at least two of thefunctional chemical groups. The candidate agents often comprise cyclicalcarbon or heterocyclic structures and/or aromatic or polyaromaticstructures substituted with one or more of the above functional groups.Candidate agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

Competitive screening assays can be done by combining KSP and a drugcandidate in a first sample. A second sample may be made combining acompound of the invention, KSP and a drug candidate. This may beperformed in either the presence or absence of microtubules. The bindingof the drug candidate is determined for both samples, and a change ordifference in binding between the two samples indicates the presence ofan agent capable of binding to KSP and potentially modulating itsactivity. That is, if the binding of the drug candidate is different inthe second sample relative to the first sample, the drug candidate iscapable of binding to KSP.

In a particularly suitable embodiment, the binding of the candidateagent is determined through the use of competitive binding assays. Inthis embodiment, the competitor is a binding moiety known to bind toKSP, such as an antibody, peptide, binding partner, ligand, etc. Undercertain circumstances, there may be competitive binding as between thecandidate agent and the binding moiety, with the binding moietydisplacing the candidate agent.

In one embodiment, the candidate agent is labeled. Either the candidateagent, or the competitor, or both, is added first to KSP for a timesufficient to allow binding, if present. Incubations can be performed atany temperature that facilitates optimal activity, typically between 4and 40° C. Incubation periods are selected for optimum activity, but mayalso be optimized to facilitate rapid high throughput screening.Typically between 0.1 and 1 hour will be sufficient. Excess reagent isgenerally removed or washed away. The second component is then added,and the presence or absence of the labeled component is followed, toindicate binding.

In a particularly suitable embodiment, the competitor is added first,followed by the candidate agent. Displacement of the competitor is anindication the candidate agent is binding to KSP and thus is capable ofbinding to, and potentially modulating, the activity of KSP. In thisembodiment, either component can be labeled. Thus, for example, if thecompetitor is labeled, the presence of label in the wash solutionindicates displacement by the agent. Alternatively, if the candidateagent is labeled, the presence of the label on the support indicatesdisplacement.

In an alternative embodiment, the candidate agent is added first, withincubation and washing, followed by the competitor. The absence ofbinding by the competitor may indicate the candidate agent is bound toKSP with a higher affinity. Thus, if the candidate agent is labeled, thepresence of the label on the support, coupled with a lack of competitorbinding, may indicate the candidate agent is capable of binding to KSP.

It may be of value to identify the binding site of KSP. This can be donein a variety of ways. In one embodiment, once KSP has been identified asbinding to the compound, KSP is fragmented or modified and the assaysrepeated to identify the necessary components for binding.

Modulation is tested by screening for candidate agents capable ofmodulating the activity of KSP comprising the steps of combining acandidate agent with KSP, as above, and determining an alteration in thebiological activity of KSP. Thus, in this embodiment, the candidateagent should both bind to KSP (although this may not be necessary), andalter its biological or biochemical activity as defined herein. Themethods include both in vitro screening methods and in vivo screening ofcells for alterations in cell cycle distribution, cell viability, or forthe presence, morpohology, activity, distribution, or amount of mitoticspindles, as are generally outlined above.

Alternatively, differential screening may be used to identify drugcandidates that bind to the native KSP, but cannot bind to modified KSP.

Positive controls and negative controls may be used in the assays.Preferably all control and test samples are performed in at leasttriplicate to obtain statistically significant results. Incubation ofall samples is for a time sufficient for the binding of the agent to theprotein. Following incubation, all samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples may be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents can be included in the screening assays.These include reagents like salts, neutral proteins, e.g., albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

Formulation and Administration

The compounds, pharmaceutically acceptable salts and solvates of FormulaI and II are administered at a therapeutically effective dosage, e.g., adosage sufficient to provide treatment for the disease states previouslydescribed. Human dosage levels are typically determined by escalatingdose ranging studies conducted in accordance with current Good ClinicalPractice, FDA and local guidelines. The amount of active compoundadministered will, of course, be dependent on the subject and diseasestate being treated, the severity of the affliction, the manner andschedule of administration and the judgment of the prescribingphysician.

The administration of the compounds and pharmaceutical formulations ofthe present invention can be done in a variety of ways, including, butnot limited to, orally, subcutaneously, intravenously, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, or intraocularly. In some instances, for example,in the treatment of wounds and inflammation, the compound or compositionmay be directly applied as a solution or spray.

Pharmaceutical formulations include a compound of Formula I or II or apharmaceutically acceptable salt or solvate thereof, and one or morepharmaceutically acceptable excipients. As is known in the art,pharmaceutical excipients are secondary ingredients that function toenable or enhance the delivery of a drug or medicine in a variety ofdosage forms (e.g.: oral forms such as tablets, capsules, and liquids;topical forms such as dermal, opthalmic, and otic forms; suppositories;injectables; respiratory forms and the like). Pharmaceutical excipientsinclude inert or inactive ingredients, synergists or chemicals thatsubstantively contribute to the medicinal effects of the activeingredient. For example, pharmaceutical excipients may function toimprove flow characteristics, product uniformity, stability, taste, orappearance, to ease handling and administration of dose, for convenienceof use, or to control bioavailability. While pharmaceutical excipientsare commonly described as being inert or inactive, it is appreciated inthe art that there is a relationship between the properties of thepharmaceutical excipients and the dosage forms containing them.

Pharmaceutical excipients suitable for use as carriers or diluents arewell known in the art, and may be used in a variety of formulations.See, e.g., Remington's Pharmaceutical Sciences, 18th Edition, A. R.Gennaro, Editor, Mack Publishing Company (1990); Remington: The Scienceand Practice of Pharmacy, 20th Edition, A. R. Gennaro, Editor,Lippincott Williams & Wilkins (2000); Handbook of PharmaceuticalExcipients, 3rd Edition, A. H. Kibbe, Editor, American PharmaceuticalAssociation, and Pharmaceutical Press (2000); and Handbook ofPharmaceutical Additives, compiled by Michael and Irene Ash, Gower(1995). The concentration of a therapeutically active agent in aformulation can vary widely, from about 0.1 to 99.9 wt.%, depending onthe nature of the formulation.

Oral solid dosage forms such as tablets will typically comprise one ormore pharmaceutical excipients, which may for example help impartsatisfactory processing and compression characteristics, or provideadditional desirable physical characteristics to the tablet. Suchpharmaceutical excipients may be selected from diluents, binders,glidants, lubricants, disintegrants, colorants, flavorants, sweeteningagents, polymers, waxes or other solubility-modulating materials.

Dosage forms for parenteral administration will generally comprisefluids, particularly intravenous fluids, i.e., sterile solutions ofsimple chemicals such as sugars, amino acids or electrolytes, which canbe easily carried by the circulatory system and assimilated. Such fluidsare typically prepared with water for injection USP. Fluids usedcommonly for intravenous (IV) use are disclosed in Remington, TheScience and Practice of Pharmacy [full citation previously provided],and include:

-   -   alcohol, e.g., 5% alcohol (e.g., in dextrose and water (“D/W”)        or D/W in normal saline solution (“NSS”), including in 5%        dextrose and water (“D5/W”), or D5/W in NSS);    -   synthetic amino acid such as Aminosyn, FreAmine, Travasol, e.g.,        3.5 or 7; 8.5; 3.5, 5.5 or 8.5% respectively;    -   ammonium chloride e.g., 2.14%;    -   dextran 40, in NSS e.g., 10% or in D5/W e.g., 10%;    -   dextran 70, in NSS e.g., 6% or in D5/W e.g., 6%;    -   dextrose (glucose, D5/W) e.g., 2.5-50%;        -   dextrose and sodium chloride e.g., 5-20% dextrose and            0.22-0.9% NaCl;    -   lactated Ringer's (Hartmann's) e.g., NaCl 0.6%, KCl 0.03%, CaCl₂        0.02%;    -   lactate 0.3%;    -   mannitol e.g., 5%, optionally in combination with dextrose e.g.,        10% or NaCl e.g., 15 or 20%;    -   multiple electrolyte solutions with varying combinations of        electrolytes, dextrose, fructose, invert sugar Ringer's e.g.,        NaCl 0.86%, KCl 0.03%, CaCl₂ 0.033%;    -   sodium bicarbonate e.g., 5%;    -   sodium chloride e.g., 0.45, 0.9, 3, or 5%;    -   sodium lactate e.g., 1/6 M; and    -   sterile water for injection        The pH of such IV fluids may vary, and will typically be from        3.5 to 8 as known in the art.

The compounds, pharmaceutically acceptable salts and solvates of theinvention can be administered alone or in combination with othertreatments, i.e., radiation, or other therapeutic agents, such as thetaxane class of agents that appear to act on microtubule formation orthe camptothecin class of topoisomerase I inhibitors. When so-used,other therapeutic agents can be administered before, concurrently(whether in separate dosage forms or in a combined dosage form), orafter administration of an active agent of the present invention.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.

EXAMPLES Example 1N-(3-amino-propyl)-N-[1-(3-benzyl-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-2-yl)-2-methyl-propyl]-4-methyl-benzamide

1A. Formula 106 where R¹, R² R⁴, R⁶, R⁷, R⁸ and R⁹ are H; R³ is Chloro;and R⁵ is Benzyl: To a solution of Formula 101,4-tert-butoxycarbonylamino-2-(9H-fluoren-9-ylmethoxycarbonylamino)-butyricacid (1.00 g, 2.27 mmol) in 20 mL of anhydrous THF was added anhydrousN-methylmorpholine (274 μL, 2.50 mmol). After the mixture was stirred inan ice-bath for 1 minute, isobutyl chloroformate (324 μL, 2.50 mmol) wasadded dropwise over 15 minutes at 0° C. The mixture was stirred in theice-bath for 1 hour, after which 4-chloro-anthranilic acid (389 mg, 2.27mmol) in 2 mL of THF was added, affording the corresponding compound ofFormula 103,2-[4-tert-butyxycarbonylamino-2-(9H-fluoren-9-ylmethoxy-carbonylamino)-butyrylamino]-4-chloro-benzoicacid, following 2 hours of additional stirring at 0° C. To the stirringcompound of Formula 103, anhydrous N-methylmorpholine (274 μL, 2.50mmol) was added and the mixture stirred overnight while slowly warmingto room temperature. The mixture was then cooled to 0° C. and treatedwith anhydrous N-methylmorpholine (274 μL, 2.50 mmol) and isobutylchloroformate (324 μL, 2.50 mmol). This was followed by addition ofbenzylamine (992 μL, 3.92 mmol) in four equal portions at roomtemperature. Once the starting material was consumed, the solvents wereevaporated and the residue partitioned between DCM and saturated sodiumbicarbonate. The organic layer was dried over sodium sulfate and thesolvent was evaporated, affording a mixture of the correspondingcompounds of Formula 104 and Formula 105,[1-(2-benzylcarbamoyl-5-chloro-phenyl-carbamoyl)-3-tert-butoxycarbonylamino-propyl]-carbamicacid 9H-fluoren-9-ylmethyl ester and[1-(3-benzyl-7-chloro-4-oxo-c,4-3,4-dihydro-quinazolin-2-yl)-3-tert-butoxycarbonyl-amino-propyl]-carbamicacid 9H-fluoren-9-ylmethyl ester, respectively (about 6:1 by LCMS) whichwas dried under vacuum and then treated with lithium hydroxidemonohydrate (95 mg, 2.27 mmol) in 60 mL of 1,4-dioxane/ethylene glycol(2/1) under reflux for 5 hours. The reaction solution was poured into200 mL of water and the product extracted with DCM. After drying theorganic layers over sodium sulfate, the solvent was evaporated and thecrude product purified by flash silica gel chromatography (stepwisegradient 1:4, 1:2, 1:1, 2:1, 4:1 with ethyl acetate:hexanes as eluent)to give the corresponding product of Formula 106,[3-amino-3-(3-benzyl-7-chloro-4-oxo-3,4-dihydro-quinazolin-2-yl)-propyl]-carbamicacid tert-butyl ester (700 mg, 70%).

1B. Formula 107 where R¹, R², R⁴, R⁶, R⁷, R⁸ and R⁹ are H; R³ is Chloro;R⁵ is Benzyl; and R¹⁰ is p-Methyl-benzyl: To a solution of[3-amino-3-(3-benzyl-7-chloro-4-oxo-3,4-dihydro-quinazolin-2-yl)-propyl]-carbamicacid tert-butyl ester (700 mg, 1.58 mmol) and DIEA (275 μL, 2.37 mmol)in 50 mL of DCM was added p-tolualdehyde (188 [2L, 1.58 mmol). Themixture was stirred for 1 hour, after which sodium triacetoxyborohydride(500 mg, 2.37 mmol) was added. After stirring an additional 3 hours, themixture was washed with saturated sodium bicarbonate solution, driedover sodium sulfate. The solvents were evaporated to dryness and theresidue was purified by flash silica gel chromatography (stepwisegradient 1:4, 1:2, 1:1, 2:1, 4:1 with ethyl acetate:hexanes as eluent)to give the corresponding, pure compound of Formula 107,[3-(3-benzyl-7-chloro-4-oxo-3,4-dihydro-quinazolin-2-yl)-3-(4-methyl-benzylamino)-propyl]-carbamicacid tert-butyl ester (760 mg, 88%).

1C. Formula 108 where R¹, R², R⁴, R⁶, R⁷, R⁸ and R⁹ are H; R³ is Chloro;R⁵ is Benzyl; R¹⁰ is p-Methyl-benzyl; and R′″ is NHBoc-Ethyl:[3-(3-Benzyl-7-chloro-4-oxo-3,4-dihydro-quinazolin-2-yl)-3-(4-methyl-benzylamino)-propyl]-carbamicacid tert-butyl ester (360 mg, 0.658 mmol) was dissolved in 10 mL ofTFA/H₂O (95/5) solution and stirred for 30 minutes. The solvents wereevaporated and the residue partitioned between DCM and saturated sodiumbicarbonate solution. The organic layer was dried over sodium sulfateand the solvents evaporated to dryness. The resulting residue, DIEA (168μL, 0.966 mmol), tert-butyl N-(2-oxoethyl) carbamate (122 mg, 0.767mmol) and sodium triacetoxyborohydride (318 mg, 0.966 mmol) were mixedin 50 mL of DCM and stirred for 1 hour. The solution was then washedwith 100 mL of saturated sodium bicarbonate solution and dried oversodium sulfate. The solvent was evaporated to dryness to give thecorresponding compound of Formula 108,{2-[3-(3-benzyl-7-chloro-4-oxo-3,4-dihydro-quinazolin-2-yl)-3-(4-methyl-benzylamino)-propylamino]-ethyl}-carbamicacid tert-butyl ester (380 mg, 98%).

1D. Formula I where R¹, R², R⁴, R⁶, R⁷, R⁸ and R⁹ are H; R³ is Chloro;R⁵ is Benzyl; R¹⁰ is p-Methyl-benzyl; and V is NR′ where R′″ isAmino-ethyl: To a solution of crude{2-[3-(3-benzyl-7-chloro-4-oxo-3,4-dihydro-quinazolin-2-yl)-3-(4-methyl-benzylamino)-propylamino]-ethyl}-carbamicacid tert-butyl ester (380 mg, 0.644 mmol) and DIEA (167 μL, 0.966 mmol)in 50 mL of DCM was added carbonyldiimidazole (157 mg, 0.966 mmol) andthe reaction mixture stirred for 1 hour. The solvent was evaporated andthe residue purified by flash silica gel chromatography (stepwisegradient 1:4, 1:2, 1:1, 2:1, 4:1 with ethyl acetate:hexanes as eluent)to give the corresponding, pure R′″-Boc-protected precursor to Formula I(250 mg, 63%). This product (250 mg, 0.405 mmol) was dissolved in 10 mLof TFA/H₂O (95/5) solution, stirred for 1 hour, and then evaporated todryness. The residue was partitioned between DCM and saturated sodiumbicarbonate, the organic layer was dried over sodium sulfate, and thesolvents evaporated to dryness. The residue was dried under vacuum for afew hours to give the desired product of Formula I,2-[-1-(2-amino-ethyl)-3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3-benzyl-7-chloro-3H-quinazolin-4-one(188 mg, 90%).

Example 2 Other Compounds of Formulae I and II

2A. Formula I where R¹, R², R⁴, R⁶, R⁸ and R⁹ are H; R³ is Chloro; R⁵ isBenzyl; R⁷ is i-Propyl; R¹⁰ is p-Methyl-benzyl; and V is NR′ where R′″is Amino-ethyl: By following the procedure described in Example 1 andsubstituting4-tert-butoxycarbonylamino-2-(9H-fluoren-9-ylmethoxycarbonylamino)-butyricacid with3-(tert-butoxycarbonylamino-methyl)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-4-methyl-pentanoicacid, there is obtained2-[-1-(2-amino-ethyl)-5-isopropyl-3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3-benzyl-7-chloro-3H-quinazolin-4-one.

2B. Formula I where R¹, R⁴, R⁶, R⁷, R⁸ and R⁹ are H; R² and R³ areMethoxy; R⁵ is Benzyl; R¹⁰ is p-Methyl-benzyl; and V is NR′ where R′″ isAmino-ethyl: By following the procedure described in Example 1 andsubstituting 4-chloro-anthranilic acid with 2-amino-4,5-dimethoxybenzoic acid, there is obtained2-[-1-(2-amino-ethyl)-3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3-benzyl-6,7-dimethoxy-3H-quinazolin-4-one.

2C. Formula I where R¹, R², R⁴, R⁶, R⁷, R⁸ and R⁹ are H; R³ is Methoxy;R⁵ is Benzyl; R¹⁰ is p-Methyl-benzyl; and V is NR′″ where R′″ isIsopropyl: By following the procedure described in Example 1 andsubstituting tert-butyl N-(2-oxoethyl) carbamate with2-methylpriopionaldehyde, there is obtained2-[-1-isopropyl-3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3-benzyl-7-chloro-3H-quinazolin-4-one.

2D. Formula II where T is Methylene; W, X, Y and Z are —C═; R¹, R², R⁴,R⁶, R⁸ and R⁹ are H; R³ is Chloro; R⁵ is Benzyl; R⁷ is i-Propyl; R¹⁰ isp-Methyl-benzyl; and V is NR′ where R′″ is Amino-ethyl: By following theprocedure described in Example 1 and substituting4-tert-butoxycarbonylamino-2-(9H-fluoren-9-ylmethoxycarbonylamino)-butyricacid with4-(tert-butoxycarbonylamino-methyl)-3-[(9H-fluoren-9-yl)-methoxycarbonyl-amino]-5-methyl-hexanoicacid, there is obtained2-[-1-(2-amino-ethyl)-5-isopropyl-3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-ylmethyl]-3-benzyl-7-chloro-3H-quinazolin-4-one.

2E. Formula II where T is Carboxyethylene; W, X, Y and Z are —C═; R¹,R², R⁴, R⁶, R⁸ and R⁹ are H; R³ is Chloro; R⁵ is Benzyl; R¹⁰ isp-Methyl-benzyl; and V is NR′ where R′″ is Isopropyl: By following theprocedure described in Example 1, substituting in4-tert-butoxycarbonylamino-2-(9H-fluoren-9-ylmethoxycarbonylamino)-butyricacid with6-(tert-butoxycarbonylamino)-4-[(9H-fluoren-9-yl)-methoxycarbonyl-amino]-3-oxo-hexanoicacid in Example 1A, and substituting tert-butyl N-(2-oxoethyl) carbamatewith 2-methyl-propionaldehyde in Example 1C, there is obtained3-benzyl-7-chloro-2-{2-[1-isopropyl-3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-2-oxo-ethyl}-3H-quinazolin-4-one.

2F. Formula II where T is Carboxyethylene; W and Y are —C═; X and Z are—N═; R¹, R², R⁴, R⁶, R⁸ and R⁹ are H; R³ is Chloro; R⁵ is Benzyl; R¹⁰ isp-Methyl-benzyl; and V is NR′ where R′″ is Isopropyl: By following theprocedure described in Example 2E and additionally substituting4-chloro-anthranilic acid with 4-amino-pyridine-5-carboxylic acid inExample 1A, there is obtained3-benzyl-2-{2-[1-isopropyl-3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-2-oxo-ethyl}-3H-pyrimido[4,5-d]pyrimidin-4-one.

Example 33-Benzyl-2-(1-benzyl-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one

3A. Formula 203 where R′, R″, R⁶, R⁷, R⁸ and R⁹ are H; and R¹⁰ isBenzyl;

DL-2-Aminoadipic acid hydrate (2 g, 11 mmol) was dissolved in 2 M NaOH(11 mL, 22 mmol). Benzaldehyde (1.4 mL, 11 mmol) was dissolved in 3.0 mLof ethanol and this solution was added to the first solution. After 10minutes the mixture was cooled to 0° C. and sodium borohydride (0.13 g,3.3 mmol) was added. After 1 hour, LCMS analysis showed the reaction tobe complete. The solution was extracted 3 times with 20 mL portions ofether, cooled to 0° C., acidified to pH 2 with conc. HCl, and theresulting precipitate was filtered to afford a damp white solid. Thesolid was washed once with a minimum amount of acetonitrile (˜1 mL) andthree times with ether. The crude solid was dissolved in 55 mL ethanoland the solution was boiled overnight. The solution was evaporated toprovide 1.22 g (47% yield for 2 steps) of the lactam product of Formula203, 1-benzyl-6-oxo-piperidine-2-carboxylic acid, which was carried onwithout further purification.

3B. Formula 204 where R′, R″, R¹ , R², R⁴, R⁶, R⁷, R⁸ and R⁹ are H; R³is Chloro; and R¹⁰ is Benzyl: 1-Benzyl-6-oxo-piperidine-2-carboxylicacid (1 g, 4.3 mmol) and DIEA (0.75 mL, 4.3 mmol) were dissolved in 20mL of dichloromethane and cooled to 0° C. Isobutylchloroformate (0.59mL, 4.3 mmol) was added and the mixture was stirred for 20 minutes. MoreDIEA (1.5 mL, 8.6 mmol) was added, followed by 4-chloroanthranilic acid(0.77 g, 4.5 mmol), and the mixture was allowed to warm to roomtemperature overnight. The mixture was washed with 1 M HCl and brine,dried over MgSO₄, and evaporated to provide a yellow oily solid. Thismaterial was dissolved in 3 mL of 2 M NaOH, washed twice with ether,cooled to 0° C., and acidified with conc. HCl. A pale yellow oil formed,which solidified upon standing. This material was filtered and driedunder vacuum to provide 0.57 g (34% yield) of the benzoic acid ofFormula 204,2-[(1-benzyl-6-oxo-piperidine-2-carbonyl)-amino]-4-chloro-benzoic acid,which was carried on without further purification.

3C. Formula I where R¹, R², R⁴, R⁶, R⁷, R⁸ and R⁹ are H; R³ is Chloro;R⁵ is Benzyl, R¹⁰ is Benzyl; and V is CR′R′″ where R′ and R″ are H: To asolution of2-[(1-benzyl-6-oxo-piperidine-2-carbonyl)-amino]-4-chloro-benzoic acid(165 mg, 0.427 mmol) in 5 mL of DMF was added EDC (245 mg, 1.28 mmol)and the mixture stirred at room temperature. After 1 hour, benzylamine(140 μL, 1.28 mmol) was added, and the reaction mixture was stirred atroom temperature for an additional 3 hours. After evaporating thesolvents, the residue was dissolved in 100 mL of DCM, and washed withsaturated sodium bicarbonate and brine. The organic layer was dried oversodium sulfate and filtered. The solvent was removed to afford crude1-benzyl-6-oxo-piperidine-2-carboxylic acid(2-benzylcarbamoyl-5-chloro-phenyl)-amide, a compound of Formula 402.This was dried under vacuum and then added to a mixture of 10 mL ofethylene glycol and sodium hydroxide (17 mg, 0.425 mmol), followed bystirring at 130° C. for two days with monitoring by LC-MS. The mixturewas then poured into 100 mL of water. After extraction with DCM, theresulting crude product was purified over silica gel (stepwise gradient1:4, 1:2, 1:1, 2:1, 4:1 with ethyl acetate:hexanes as eluent) to givethe desired pure product of Formula I,3-benzyl-2-(1-benzyl-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one(20 mg,10%).

Example 4 Other Compounds of Formulae I and II

4A. Varying T, V and R⁶ to R⁹ By following the procedure described inExample 3 and substituting DL-2-aminoadipic acid hydrate with thefollowing:

-   -   2-amino-pentanedioic acid;    -   4-amino-2,2-dimethyl-pentanedioic acid;    -   2-amino-3-methyl-hexanedioic acid;    -   2-amino-4-isopropyl-hexanedioic acid;    -   2-amino-3,4-dihydroxy-hexanedioic acid;    -   5-amino-2,3-diethoxy-hexanedioic acid;    -   4-amino-octanedioic acid; and    -   5-amino-6-carboxymethoxy-hexanoic acid,        there are obtained the following respective compounds:    -   3-benzyl-2-(1-benzyl-5-oxo-pyrrolidin-2-yl)-7-chloro-3H-quinazolin-4-one;    -   3-benzyl-2-(1-benzyl-4,4-dimethyl-5-oxo-pyrrolidin-2-yl)-7-chloro-3H-quinazolin-4-one;    -   3-benzyl-2-(1-benzyl-3-methyl-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one;    -   3-benzyl-2-(1-benzyl-4-isopropyl-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one;    -   3-benzyl-2-(1-benzyl-3,4-dihydroxy-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one;    -   3-benzyl-2-(1-benzyl-4,5-diethoxy-6-oxo-piperidin-2-yl)-7-chloro-3H-quinazolin-4-one;    -   3-benzyl-2-[3-(1-benzyl-5-oxo-pyrrolidin-2-yl)-propyl]-7-chloro-3H-quinazolin-4-one    -   3-benzyl-2-(1-benzyl-6-oxo-piperidin-2-ylmethoxymethyl)-7-chloro-3H-quinazolin-4-one.

4B. Varying R¹⁰ By following the procedure described in Examples 3 and4A, and substituting benzaldeyhde with 4-methyl-benzaldehyde, there areobtained:

-   -   3-benzyl-2-[1-(4-methyl-benzyl)-5-oxo-pyrrolidin-2-yl]-7-chloro-3H-quinazolin-4-one;    -   3-benzyl-2-[4,4-dimethyl-1-(4-methyl-benzyl)-5-oxo-pyrrolidin-2-yl]-7-chloro-3H-quinazolin-4-one;    -   3-benzyl-2-[3-methyl-1-(4-methyl-benzyl)-6-oxo-piperidin-2-yl]-7-chloro-3H-quinazolin-4-one;    -   3-benzyl-2-[4-isopropyl-1-(4-methyl-benzyl)-6-oxo-piperidin-2-yl]-7-chloro-3H-quinazolin-4-one;    -   3-benzyl-2-[3,4-dihydroxy-1-(4-methyl-benzyl)-6-oxo-piperidin-2-yl]-7-chloro-3H-quinazolin-4-one;    -   3-benzyl-2-[4,5-diethoxy-1-(4-methyl-benzyl)-6-oxo-piperidin-2-yl]-7-chloro-3H-quinazolin-4-one;    -   3-benzyl-2-{3-[1-(4-methyl-benzyl)-5-oxo-pyrrolidin-2-yl]-propyl}-7-chloro-3H-quinazolin-4-one;        and    -   3-benzyl-2-[1-(4-methyl-benzyl)-6-oxo-piperidin-2-ylmethoxymethyl]-7-chloro-3H-quinazolin-4-one;

4C. Varying T, V, and R′ or R⁹ By following the procedure described inExample 3, substituting DL-2-aminoadipic acid hydrate with thefollowing:

-   -   2-amino-4-(2-tert-butoxycarbonylamino-ethyl)-pentanedioic acid;    -   2-amino-5-(2-tert-butoxycarbonylamino-ethyl)-hexanedioic acid;        and    -   5-amino-2-(2-tert-butoxycarbonylamino-ethyl)-6-(tert-butoxycarbonyl-carboxymethyl-amino)-hexanoic        acid,        and de-protecting the product thus-obtained (for example, as        described in the second part of Example 1D), there are obtained        the following respective compounds:    -   3-benzyl-2-[4-(2-amino-ethyl)-1-benzyl-5-oxo-pyrrolidin-2-yl]-7-chloro-3H-quinazolin-4-one;    -   3-benzyl-2-[5-(2-amino-ethyl)-1-benzyl-6-oxo-piperidin-2-yl]-7-chloro-3H-quinazolin-4one;        and    -   2-({[5-(2-amino-ethyl)-1-benzyl-6-oxo-piperidin-2-ylmethyl]-amino}-methyl)-3-benzyl-7-chloro-3H-quinazolin-4-one.

4D. Varying T, V, R′, R⁹ and R¹⁰ By following the procedure described inExample 4C and substituting benzaldeyhde with (2-oxo-ethyl)-carbamicacid tert-butyl ester, there are obtained:

-   -   3-benzyl-2-[1,4-bis-(2-amino-ethyl)-5-oxo-pyrrolidin-2-yl]-7-chloro-3H-quinazolin-4-one;    -   3-benzyl-2-[1,5-bis-(2-amino-ethyl)-6-oxo-piperidin-2-yl]-7-chloro-3H-quinazolin-4-one;        and    -   3-benzyl-2-({[1,5-bis-(2-amino-ethyl)-6-oxo-piperidin-2-ylmethyl]-amino}-methyl)-7-chloro-3H-quinazolin-4-one.

4E. Varying T, V, R¹to R⁴, R⁶ to R⁹ and W, X, Y and Z By following theprocedure as described with 2-amino-pentanedioic acid,4-amino-2,2-dimethyl-pentanedioic acid and 2-amino-3-methyl-hexanedioicacid in Example 4A, and additionally substituting 4-chloroanthranilicacid (from Example 3B) with the following:

-   -   3-amino-pyrazine-2-carboxylic acid;    -   3-amino-1,4-dihydro-pyridine-2-carboxylic acid;    -   2-amino-cyclopent-1-enecarboxylic acid;    -   4-amino-2,5-dihydro-furan-3-carboxylic acid; and    -   3-amino-1H-pyrrole-2-carboxylic acid,        there are obtained the following respective compounds:    -   3-benzyl-2-(1-benzyl-5-oxo-pyrrolidin-2-yl)-3H-pteridin-4-one;    -   3-benzyl-2-(1-benzyl-5-oxo-pyrrolidin-2-yl)-5,8-dihydro-3H-pyrido[3,2-d]pyrimidin-4-one;    -   3-benzyl-2-(1-benzyl-5-oxo-pyrrolidin-2-yl)-3,5,6,7-tetrahydro-cyclopentapyrimidin-4-one;    -   3-benzyl-2-(1-benzyl-5-oxo-pyrrolidin-2-yl]-5,7-dihydro-3H-furo[3,4-d]pyrimidin-4-one;    -   3-benzyl-2-(1-benzyl-5-oxo-pyrrolidin-2-yl]-3,7-dihydro-pyrrolo[3,2-d]pyrimidin-4-one;    -   3-benzyl-2-(1-benzyl-4,4-dimethyl-5-oxo-pyrrolidin-2-yl)-3H-pteridin-4-one;    -   3-benzyl-2-(1-benzyl-4,4-dimethyl-5-oxo-pyrrolidin-2-yl)-5,8-dihydro-3H-pyrido[3,2-d]pyrimidin-4-one;    -   3-benzyl-2-(1-benzyl-4,4-dimethyl-5-oxo-pyrrolidin-2-yl)-3,5,6,7-tetrahydro-cyclopentapyrimidin-4-one;    -   3-benzyl-2-(1-benzyl-4,4-dimethyl-5-oxo-pyrrolidin-2-yl]-5,7-dihydro-3H-furo[3,4-d]pyrimidin-4-one;    -   3-benzyl-2-(1-benzyl-4,4-dimethyl-5-oxo-pyrrolidin-2-yl]-3,7-dihydro-pyrrolo[3,2-d]pyrimidin-4-one;    -   3-benzyl-2-(1-benzyl-3-methyl-6-oxo-piperidin-2-yl)-3H-pteridin-4-one;    -   3-benzyl-2-(1-benzyl-3-methyl-6-oxo-piperidin-2-yl)-5,8-dihydro-3H-pyrido[3,2-d]pyrimidin-4-one;    -   3-benzyl-2-(1-benzyl-3-methyl-6-oxo-piperidin-2-yl)-3,5,6,7-tetrahydro-cyclopentapyrimidin-4-one;    -   3-benzyl-2-(1-benzyl-3-methyl-6-oxo-piperidin-2-yl]-5,7-dihydro-3H-furo[3,4-d]pyrimidin-4-one;        and    -   3-benzyl-2-(1-benzyl-3-methyl-6-oxo-piperidin-2-yl]-3,7-dihydro-pyrrolo[3,2-d]pyrimidin-4-one.

Example 53-Benzyl-7-chloro-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3H-quinazolin-4-one

5A. Formula 302 where R⁶, R⁷, R⁸ and R⁹ are H: DL-2,4-Diaminobutyricacid (4.9 g, 26 mmol) and sodium bicarbonate (6.5 g, 77 mmol) wasdissolved in 25 mL of water. Copper sulfate (3.2 g, 13 mmol) wasdissolved in 25 mL of water and this solution was added to the firstsolution. To this mixture was added a solution of di-(tert-butyl)pyrocarbonate (7.3 g, 33.5 mmol) dissolved in 32 mL of acetone. Afterstirring for 24 hours, methanol was added to the mixture and stirringwas continued for another 18 hours. The resulting light blue precipitatewas filtered, washed twice with water, and dried under vacuum to provide3.3 g (51%) of the mono-Boc-protected copper complex of diaminobutyricacid. This copper complex (3.3 g, 6.7 mmol) was suspended in 300 mL ofwater and quinolinol (2.5 g, 17 mmol) was added. After 5 hours, thesuspension was filtered off and the liquid was evaporated to provideapproximately 3.5 g of a wet yellow solid. This material was dissolvedin 200 mL of 30% methanol in benzene. To this solution was addeddropwise (trimethylsilyl)diazomethane (2.0 M in hexanes, approximately12 mL, approximately 24 mmol) until a deep yellow color persisted andbubbling ceased. This solution was stirred for 1 hour. Acetic acid wasadded dropwise until the deep yellow color was discharged and bubblingceased, and then the mixture was evaporated. The material was purifiedtwice by flash chromatography (EtOAc to 5% MeOH in EtOAc) to provide themethyl ester of Formula 302, 2-amino-4-tert-butoxycarbonylamino-butyricacid methyl ester, as a pale yellow wax (1.4 g, 58%).

5B. Formula 303 where R⁶, R⁷, R⁸ and R⁹ are H; and R¹⁰ isp-Methyl-benzyl:

To a solution of 2-amino-4-tert-butoxycarbonylamino-butyric acid methylester (0.60 g, 2.58 mmol) in 100 mL of DCM was added p-tolualdehyde (282μL, 2.39 mmol) and the mixture stirred at room temperature for 1 hour.Sodium triacetoxyborohydride (820 mg, 3.87 mmol) was added and themixture stirred overnight. The solution was then washed with water anddried over magnesium sulfate. The solvents were evaporated and theresidue dissolved in 100 mL of 2M HCl in dioxane solution and stirredfor 2 hours. The solvent was evaporated and the residue dried undervacuum to give the desired compound of Formula 303,4-amino-2-(4-methyl-benzylamino)-butyric acid methyl ester as its HClsalt (475 mg, 62%). This was taken on without further purification.

5C. Formula 304 where R⁶ , R⁷, R⁸ and R⁹ are H; and R¹⁰ isp-Methyl-benzyl:

To a solution of 4-amino-2-(4-methyl-benzylamino)-butyric acid methylester HCl salt (923 mg, 3.13 mmol) and DIEA (1.08 mL, 6.26 mmol) in 100mL of DCM was added carbonyldiimidazole (760 mg, 4.69 mmol). Thereaction mixture was stirred for 1 hour, after which the solvents wereevaporated. The residue was dissolved in 20 mL of MeOH:H₂O (2:1)solution to which was added LiOH (150 mg, 6.26 mmol). The mixture wasstirred at room temperature for 3 hours and then adjusted to pH ˜7 byadding Dowex-H+ resin. The solution was filtered, the solvent wasevaporated, and the residue dried under vacuum to give the desiredcompound of Formula 304,3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidine-4-carboxylic acid (520mg, 67%), which was taken on without further purification.

5D. Formula 402 where R¹, R², R⁴, R⁶, R⁷, R⁸ and R⁹ are H; R⁵ is Benzyl;R¹⁰ is p-Methyl-benzyl; and V is N—R′″ where R′″ is H: To a solution of3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidine-4-carboxylic acid (400mg, 1.61 mmol) in 20 mL of DMF was added anhydrous N-methylmorpholine(212 μL, 1.93 mmol). After cooling in an ice-bath for 10 min, isobutylchloroformate (251 μL, 1.93 mmol) was added dropwise at whilemaintaining the temperature below 5° C. The mixture was stirred in theice-bath for 1 hour, and 4-chloroanthranilic acid (331 mg, 1.93 mmol) in1 mL of DMF was added. The mixture was stirred an additional 5 h duringwhich the temperature was allowed to warm to room temperature to affordthe corresponding intermediate product of Formula 305, which was carriedon without isolation or purification. EDC (618 mg, 3.22 mmol) was thenadded into the reaction, the mixture was stirred for 1 hour, andbenzylamine (528 μL, 4.83 mmol) was added. The resulting solution wasstirred overnight, after which the solvents were evaporated and theresidue purified by flash silica gel chromatography (stepwise gradient1:4, 1:2, 1:1, 2:1, 4:1 with ethyl acetate:hexanes as eluent) to givethe pure bis-amide product of Formula 402,3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidine-4-carboxylic acid(2-benzylcarbamoyl-5-chloro-phenyl)-amide (430 mg, 54%).

5E. Formula I where R¹, R², R⁴, R⁶, R⁷, R⁸ and R⁹ are H; R³ is Chloro;R⁵ is Benzyl; R¹⁰ is p-Methyl-benzyl; and V is N—R′″ where R′″ is H:3-(4-Methyl-benzyl)-2-oxo-hexahydro-pyrimidine-4-carboxylic acid(2-benzylcarbamoyl-5-chloro-phenyl)-amide (350 mg, 0.713 mmol) wasdissolved in 60 mL of ethylene glycol to which was added sodiumhydroxide (60 mg, 1.5 mmol). The mixture was stirred at 140° C. for 20hours and monitored by LC-MS. Following consumption of startingmaterial, the reaction mixture was poured into 100 mL of water. Afterextraction with DCM, the crude product was purified by flash silica gelchromatography (stepwise gradient 1:4, 1:2, 1:1, 2:1, 4:1 with ethylacetate:hexanes as eluent) to give the desired product of Formula I,3-benzyl-7-chloro-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3H-quinazolin-4-one(180 mg, 53%).

Example 6 Compounds of Formula II where R⁶ to R⁹ are H; R⁵ is Benzyl;R¹⁰ is p-Methyl-benzyl; T is Methylene and V is N—R′″ where R′″ is H,Varying R¹ to R⁴ and W, X, Y and Z

By following the procedure as described in Example 5 and substituting4-chloroanthranilic acid with the following:

-   -   3-amino-pyrazine-2-carboxylic acid;    -   3-amino-1,4-dihydro-pyridine-2-carboxylic acid;    -   2-amino-cyclopent-1-enecarboxylic acid;    -   4-amino-2,5-dihydro-furan-3-carboxylic acid; and    -   3-amino-1H-pyrrole-2-carboxylic acid,        there are obtained the following respective compounds:    -   3-benzyl-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3H-pteridin-4-one;    -   3-benzyl-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-5,8-dihydro-3H-pyrido[3,2-d]pyrimidin-4-one;    -   3-benzyl-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3,5,6,7-tetrahydro-cyclopentapyrimidin-4-one;    -   3-benzyl-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-5,7-dihydro-3H-furo[3,4-d]pyrimidin-4-one;        and    -   3-benzyl-2-[3-(4-methyl-benzyl)-2-oxo-hexahydro-pyrimidin-4-yl]-3,7-dihydro-pyrrolo[3,2-d]pyrimidin-4-one.

Example 7 Induction of Mitotic Arrest in Cell Populations Treated with aKSP Inhibitor

FACS analysis to determine cell cycle stage by measuring DNA content isperformed as follows. Skov-3 cells (human ovarian cancer) are split 1:10for plating in 10 cm dishes and grown to subconfluence with RPMI 1640medium containing 5% fetal bovine serum (FBS). The cells are thentreated with either 10 nM paclitaxel, 400 nM test compound, 200 nM testcompound, or 0.25% DMSO (vehicle for compounds) for 24 hours. A wellknown anti-mitotic agent, such as placitaxel, is used as a positivecontrol. Cells are then rinsed off the plates with PBS containing 5 mMEDTA, pelleted, washed once in PBS containing 1% FCS, and then fixedovernight in 85% ethanol at 4° C. Before analysis, the cells arepelleted, washed once, and stained in a solution of 10 μg propidiumiodide and 250 μg of ribonuclease (RNAse) A per milliliter at 37° C. forhalf an hour. Flow cytometry analysis is performed on a Becton-DickinsonFACScan, and data from 10,000 cells per sample is analyzed with Modfitsoftware.

Monopolar Spindle Formation Following Application of a Quinazolinone KSPInhibitor

To determine the nature of G2/M accumulation, human tumor cell linesSkov-3 (ovarian), HeLa (cervical), and A549 (lung) are plated in 96-wellplates at densities of 4,000 cells per well (SKOV-3 & HeLa) or 8,000cells per well (A549), allowed to adhere for 24 hours, and treated withvarious concentrations of the test compounds for 24 hours. Cells arefixed in 4% formaldehyde and stained with antitubulin antibodies(subsequently recognized using fluorescently-labeled secondary antibody)and Hoechst dye (which stains DNA). The cells can be visually inspectedto assess the effects of the test compounds. For example, microinjectionof anti-KSP antibodies causes mitotic arrest with arrested cellsdisplaying monopolar spindles.

Example 8 Inhibition of Cellular Proliferation in Tumor Cell LinesTreated with KSP Inhibitors

Cells are plated in 96-well plates at densities from 1000-2500cells/well (depending on the cell line) and allowed to adhere/grow for24 hours. They are then treated with various concentrations of testcompound for 48 hours. The time at which compounds are added isconsidered T₀. A tetrazolium-based assay using the reagent3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) (U.S. Pat. No. 5,185,450) (see Promega product catalog #G3580,CellTiter 96® AQ_(ueous) One Solution Cell Proliferation Assay) is usedto determine the number of viable cells at T₀ and the number of cellsremaining after 48 hours compound exposure. The number of cellsremaining after 48 hours is compared to the number of viable cells atthe time of test compound addition, allowing for calculation of growthinhibition. The growth over 48 hours of cells in control wells treatedwith vehicle only (0.25% DMSO) is considered 100% growth and the growthof cells in wells with compounds is compared to this. Active KSPinhibitors inhibit cell proliferation in one or more human tumor celllines of the following tumor types: lung (NCI-H460, A549), breast(MDA-MB-231, MCF-7, MCF-7/ADR-RES), colon (HT29, HCT15), ovarian(SKOV-3, OVCAR-3), leukemia (HL-60(TB), K-562), central nervous system(SF-268), renal (A498), osteosarcoma (U2-OS), and cervical (HeLa), andmouse tumor line (B16, melanoma).

Calculation Of GI₅₀: A GI₅₀ is calculated by plotting the concentrationof compound in μM vs the percentage of cell growth of cell growth intreated wells. The GI₅₀ calculated for the compounds is the estimatedconcentration at which growth is inhibited by 50% compared to control,i.e., the concentration at which:100×[(Treated ₄₈ −T ₀)/(Control₄₈ −T ₀)]=50.All concentrations of compounds are tested in duplicate and controls areaveraged over 12 wells. A very similar 96-well plate layout and GI₅₀calculation scheme is used by the National Cancer Institute (see Monks,et al., J. Natl. Cancer Inst. 83:757-766 (1991)). However, the method bywhich the National Cancer Institute quantitates cell number does not useMTS, but instead employs alternative methods.

Calculation Of IC₅₀: Measurement of a compound's IC₅₀ for KSP activityuses an ATPase assay. The following solutions are used: Solution 1consists of 3 mM phosphoenolpyruvate potassium salt (Sigma P-7127), 2 mMATP (Sigma A-3377), 1 mM IDTT (Sigma D-9779), 5 μM paclitaxel (SigmaT-7402), 10 ppm antifoam 289 (Sigma A-8436), 25 mM Pipes/KOH pH 6.8(Sigma P6757), 2 mM MgC12 (VWR JT400301), and 1 mM EGTA (Sigma E3889).Solution 2 consists of 1 mM NADH (Sigma N8129), 0.2 mg/ml BSA (SigmaA7906), pyruvate kinase 7U/ml, L-lactate dehydrogenase 10 U/ml (SigmaP0294), 100 nM KSP motor domain, 50 μg/ml microtubules, 1 mM DTT (SigmaD9779), 5 μM paclitaxel (Sigma T-7402), 10 ppm antifoam 289 (SigmaA-8436), 25 mM Pipes/KOH pH 6.8 (Sigma P6757), 2 mM MgC12 (VWRJT4003-01), and 1 mM EGTA (Sigma E3889). Serial dilutions (8-12 two-folddilutions) of the composition are made in a 96-well microtiter plate(Corning Costar 3695) using Solution 1. Following serial dilution eachwell has 50 μl of Solution 1. The reaction is started by adding 50 μl ofSolution 2 to each well. This can be done with a multichannel pipettoreither manually or with automated liquid handling devices. Themicrotiter plate is then transferred to a microplate absorbance readerand multiple absorbance readings at 340 nm are taken for each well in akinetic mode. The observed rate of change, which is proportional to theATPase rate, is then plotted as a function of the compoundconcentration. For a standard IC₅₀ determination the data acquired isfit by the following four parameter equation using a nonlinear fittingprogram (e.g., Grafit 4):$y = {\frac{Range}{1 + \left( \frac{x}{{IC}_{50}} \right)^{s}} + {Background}}$where y is the observed rate and x the compound concentration.

Example 9 Inhibition of Cellular Viability in Tumor Cell Lines Treatedwith KSP Inhibitors

Materials and Solutions:

-   -   Cells: SKOV3, Ovarian Cancer (human).    -   Media: Phenol Red Free RPMI+5% Fetal Bovine Serum+2 mM        L-glutamine.    -   Colorimetric Agent for Determining Cell Viability: Promega MTS        tetrazolium compound.    -   Control Compound for max cell kill: Topotecan, 1 μM.

Procedure: Day 1—Cell Plating: Adherent SKOV3 cells are washed with 10mLs of PBS followed by the addition of 2 mLs of 0.25% trypsin andincubation for 5 minutes at 37° C. The cells are rinsed from the flaskusing 8 mL of media (phenol red-free RPMI+5% FBS) and transferred tofresh flask. Cell concentration is determined using a Coulter counterand the appropriate volume of cells to achieve 1000 cells/100μL iscalculated. 100 μL of media cell suspension (adjusted to 1000 cells/100μL) is added to all wells of 96-well plates, followed by incubation for18 to 24 hours at 37° C., 100% humidity, and 5% CO₂, allowing the cellsto adhere to the plates.

Procedure: Day 2—Compound Addition: To one column of the wells of anautoclaved assay block are added an initial 2.5 μL of test compound(s)at 400× the highest desired concentration. 1.25 μL of 400× (400 μM)Topotecan is added to other wells (ODs from these wells are used tosubtract out for background absorbance of dead cells and vehicle). 500μL of media without DMSO are added to the wells containing testcompound, and 250 μL to the Topotecan wells. 250 μL of media+0.5% DMSOis added to all remaining wells, into which the test compound(s) areserially diluted. By row, compound-containing media is replica plated(in duplicate) from the assay block to the corresponding cell plates.The cell plates are incubated for 72hours at 37° C., 100% humidity, and5% CO₂.

Procedure: Day 4—MTS Addition and OD Reading: The plates are removedfrom the incubator and 40 μl MTS/PMS is added to each well. Plates arethen incubated for 120 minutes at 37° C., 100% humidity, 5% CO₂,followed by reading the ODs at 490 nm after a 5 second shaking cycle ina ninety-six well spectrophotometer.

Data Analysis The normalized % of control (absorbance−background) iscalculated and an XLfit is used to generate a dose-response curve fromwhich the concentration of compound required to inhibit viability by 50%is determined.

The compounds of the present invention show activity when tested in oneor more of the methods described in Examples 7, 8 and 9.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. All patents and publications cited above arehereby incorporated by reference.

1. A compound selected from the group represented by Formula I:

where: V is a covalent bond, R¹, R², R³ and R⁴ are independentlyhydrogen, hydroxy, optionally substituted alkyl, optionally substitutedalkoxy, halogen or cyano; R⁵ is hydrogen, optionally substituted alkyl,optionally substituted aryl, optionally substituted aralkyl, optionallysubstituted heteroaryl, or optionally substituted heteroaralkyl; R⁶ toR⁹ are independently hydrogen, hydroxy, optionally substituted alkyl,optionally substituted alkoxy, optionally substituted aryl or optionallysubstituted alkylamino, and R¹⁰ is hydrogen, optionally substitutedalkyl, optionally substituted aryl, optionally substituted aralkyl,optionally substituted heteroaryl, or optionally substitutedheteroaralkyl, or a pharmaceutically acceptable salt or thereof.
 2. Thecompound of claim 1 wherein R¹, R², R³ and R⁴ are independentlyhydrogen, halo, lower alkyl, substituted lower alkyl, lower alkoxy orcyano.
 3. The compound of claim 2 wherein R¹, R², R³ and R⁴ areindependently hydrogen, chloro, fluoro, methyl, methoxy, or cyano. 4.The compound of claim 2 where R¹, R², R³ and R⁴ are hydrogen, or threeof R¹, R², R³ and R⁴ are hydrogen and the fourth is halo, methoxy,methyl or cyano. 5-10. (canceled)
 11. A pharmaceutical formulationcomprising a pharmaceutically acceptable excipient and an effectiveamount of a compound of claim
 1. 12-15. (canceled)
 16. A compound of thegroup represented by Formula II:

where: T is a covalent bond or optionally substituted lower alkylene; Vis a covalent bond; W, X, Y and Z are independently N, C, O, S orabsent, provided that: no more than one of W, X, Y or Z is absent, nomore than two of W, X, Y and Z are —N═, and W, X, Y or Z can be O or Sonly when one of W, X, Y or Z is absent; R¹, R², R³ and R⁴ areindependently hydrogen, hydroxy, optionally substituted alkyl,optionally substituted alkoxy, halogen or cyano, provided that R¹, R²,R³ or R⁴ is absent where W, X, Y or Z, respectively, is —N═, O, S or isabsent; R⁵ is hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl, optionally substitutedheteroaryl, and optionally substituted heteroaralkyl; R⁶ to R⁹ areindependently hydrogen, hydroxy, optionally substituted alkyl,optionally substituted alkoxy, optionally substituted aryl or optionallysubstituted alkylamino, and R¹⁰ is hydrogen, optionally substitutedalkyl, optionally substituted aryl, optionally substituted aralkyl,optionally substituted heteroaryl, or optionally substitutedheteroaralkyl, or a pharmaceutically acceptable salt thereof.
 17. Thecompound of claim 16 wherein T is optionally substituted lower alkylene.18. The compound of claim 17 wherein T is methylene. 19-23. (canceled)24. The compound of claim 1 wherein R⁵ is aralkyl or substitutedaralkyl.
 25. The compound of claim 24 wherein R⁵ is benzyl orsubstituted benzyl.
 26. The compound of claim 1 wherein R⁶ to R⁹ areindependently hydrogen or optionally substituted lower alkyl.
 27. Thecompound of claim 26 wherein R⁶ to R⁹ are hydrogen.
 28. The compound ofclaim 1 wherein R¹⁰ is optionally substituted benzyl or optionallysubstituted phenyl.
 29. The compound of claim 28 wherein R¹⁰ is benzylor p-methyl-benzyl.
 30. The compound of claim 16 wherein R¹, R², R³ andR⁴ are independently selected from hydrogen, halo, lower alkyl,substituted lower alkyl, lower alkoxy, and cyano.
 31. The compound ofclaim 30 wherein R¹, R², R³ and R⁴ are independently selected fromhydrogen, chloro, fluoro, methyl, methoxy, and cyano.
 32. The compoundof claim 30 wherein R¹, R², R³ and R⁴ are hydrogen, or three of R¹, R²,R³ and R⁴ are hydrogen and the fourth is halo, methoxy, methyl or cyano.33. The compound of claim 16 wherein R⁵ is aralkyl or substitutedaralkyl.
 34. The compound of claim 33 wherein R⁵ is benzyl orsubstituted benzyl.
 35. The compound of claim 16 wherein R⁶ to R⁹ areindependently hydrogen or optionally substituted lower alkyl.
 36. Thecompound of claim 35 wherein R⁶ to R⁹ are hydrogen.
 37. The compound ofclaim 16 wherein R¹⁰ is optionally substituted benzyl or optionallysubstituted phenyl.
 38. The compound of claim 37 wherein R¹⁰ is benzylor p-methyl-benzyl.